Publications by authors named "Klaus Uberla"

114 Publications

Upregulation of CCR4 in activated CD8+ T cells indicates enhanced lung-homing in patients with severe acute SARS-CoV-2 infection.

Eur J Immunol 2021 Mar 30. Epub 2021 Mar 30.

Department of Internal Medicine 5, Hematology and Oncology, University Hospital Erlangen and Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany.

COVID-19 is a life-threatening disease leading to bilateral pneumonia and respiratory failure. The underlying reasons why a smaller percentage of patients present with severe pulmonary symptoms whereas the majority is only mildly affected are to date not well understood. Comparing the immunological phenotype in healthy donors and patients with mild versus severe COVID-19 shows that in COVID-19 patients, NK/B cell activation and proliferation are enhanced independent of severity. As an important precondition for effective antibody responses, T follicular helper cells and antibody secreting cells are increased both in patients with mild and severe SARS-CoV-2 infection. Beyond this, T-cells in COVID-19 patients exhibit a stronger activation profile with differentiation towards effector cell phenotypes. Importantly, when looking at the rates of pulmonary complications in COVID-19 patients, the chemokine receptor CCR4 is higher expressed by both CD4 and CD8 T-cells of patients with severe COVID-19. This raises the hypothesis that CCR4 upregulation on T-cells in the pathogenesis of COVID-19 promotes stronger T-cell attraction to the lungs leading to increased immune activation with presumably higher pulmonary toxicity. Our study contributes significantly to the understanding of the immunological changes during COVID-19, as new therapeutic agents, preferentially targeting the immune system, are highly warranted. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1002/eji.202049135DOI Listing
March 2021

Complement Activation in Kidneys of Patients With COVID-19.

Front Immunol 2020;11:594849. Epub 2021 Jan 29.

Department of Nephropathology, Institute of Pathology, Friedrich-Alexander-University (FAU) Erlangen-Nürnberg, Erlangen, Germany.

Most patients who became critically ill following infection with COVID-19 develop severe acute respiratory syndrome (SARS) attributed to a maladaptive or inadequate immune response. The complement system is an important component of the innate immune system that is involved in the opsonization of viruses but also in triggering further immune cell responses. Complement activation was seen in plasma adsorber material that clogged during the treatment of critically ill patients with COVID-19. Apart from the lung, the kidney is the second most common organ affected by COVID-19. Using immunohistochemistry for complement factors C1q, MASP-2, C3c, C3d, C4d, and C5b-9 we investigated the involvement of the complement system in six kidney biopsies with acute kidney failure in different clinical settings and three kidneys from autopsy material of patients with COVID-19. Renal tissue was analyzed for signs of renal injury by detection of thrombus formation using CD61, endothelial cell rarefaction using the marker E-26 transformation specific-related gene (ERG-) and proliferation using proliferating cell nuclear antigen (PCNA)-staining. SARS-CoV-2 was detected by hybridization and immunohistochemistry. Biopsies from patients with hemolytic uremic syndrome (HUS, n = 5), severe acute tubular injury (ATI, n = 7), zero biopsies with disseminated intravascular coagulation (DIC, n = 7) and 1 year protocol biopsies from renal transplants (Ctrl, n = 7) served as controls. In the material clogging plasma adsorbers used for extracorporeal therapy of patients with COVID-19 C3 was the dominant protein but collectin 11 and MASP-2 were also identified. SARS-CoV-2 was sporadically present in varying numbers in some biopsies from patients with COVID-19. The highest frequency of CD61-positive platelets was found in peritubular capillaries and arteries of COVID-19 infected renal specimens as compared to all controls. Apart from COVID-19 specimens, MASP-2 was detected in glomeruli with DIC and ATI. In contrast, the classical pathway (i.e. C1q) was hardly seen in COVID-19 biopsies. Both C3 cleavage products C3c and C3d were strongly detected in renal arteries but also occurs in glomerular capillaries of COVID-19 biopsies, while tubular C3d was stronger than C3c in biopsies from COVID-19 patients. The membrane attack complex C5b-9, demonstrating terminal pathway activation, was predominantly deposited in COVID-19 biopsies in peritubular capillaries, renal arterioles, and tubular basement membrane with similar or even higher frequency compared to controls. In conclusion, various complement pathways were activated in COVID-19 kidneys, the lectin pathway mainly in peritubular capillaries and in part the classical pathway in renal arteries whereas the alternative pathway seem to be crucial for tubular complement activation. Therefore, activation of the complement system might be involved in the worsening of renal injury. Complement inhibition might thus be a promising treatment option to prevent deregulated activation and subsequent collateral tissue injury.
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http://dx.doi.org/10.3389/fimmu.2020.594849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878379PMC
February 2021

CRNKL1 Is a Highly Selective Regulator of Intron-Retaining HIV-1 and Cellular mRNAs.

mBio 2021 01 19;12(1). Epub 2021 Jan 19.

Institute of Clinical and Molecular Virology, University Hospital Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany

The HIV-1 Rev protein is a nuclear export factor for unspliced and incompletely spliced HIV-1 RNAs. Without Rev, these intron-retaining RNAs are trapped in the nucleus. A genome-wide screen identified nine proteins of the spliceosome, which all enhanced expression from the HIV-1 unspliced RNA after CRISPR/Cas knockdown. Depletion of DHX38, WDR70, and four proteins of the Prp19-associated complex (ISY1, BUD31, XAB2, and CRNKL1) resulted in a more than 20-fold enhancement of unspliced HIV-1 RNA levels in the cytoplasm. Targeting of CRNKL1, DHX38, and BUD31 affected nuclear export efficiencies of the HIV-1 unspliced RNA to a much larger extent than splicing. Transcriptomic analyses further revealed that CRNKL1 also suppresses cytoplasmic levels of a subset of cellular mRNAs, including some with selectively retained introns. Thus, CRNKL1-dependent nuclear retention is a novel cellular mechanism for the regulation of cytoplasmic levels of intron-retaining HIV-1 mRNAs, which HIV-1 may have harnessed to direct its complex splicing pattern. To regulate its complex splicing pattern, HIV-1 uses the adaptor protein Rev to shuttle unspliced or partially spliced mRNA from the nucleus to the cytoplasm. In the absence of Rev, these RNAs are retained in the nucleus, but it is unclear why. Here we identify cellular proteins whose depletion enhances cytoplasmic levels of the HIV-1 unspliced RNA. Depletion of one of them, CRNKL1, also increases cytoplasmic levels of a subset of intron-retaining cellular mRNA, suggesting that CRNKL1-dependent nuclear retention may be a basic cellular mechanism exploited by HIV-1.
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http://dx.doi.org/10.1128/mBio.02525-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845644PMC
January 2021

IMU-838, a Developmental DHODH Inhibitor in Phase II for Autoimmune Disease, Shows Anti-SARS-CoV-2 and Broad-Spectrum Antiviral Efficacy In Vitro.

Viruses 2020 12 5;12(12). Epub 2020 Dec 5.

Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-Nürnberg (FAU), Schlossgarten 4, 91054 Erlangen, Germany.

The ongoing pandemic spread of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) demands skillful strategies for novel drug development, drug repurposing and cotreatments, in particular focusing on existing candidates of host-directed antivirals (HDAs). The developmental drug IMU-838, currently being investigated in a phase 2b trial in patients suffering from autoimmune diseases, represents an inhibitor of human dihydroorotate dehydrogenase (DHODH) with a recently proven antiviral activity in vitro and in vivo. Here, we established an analysis system for assessing the antiviral potency of IMU-838 and DHODH-directed back-up drugs in cultured cell-based infection models. By the use of SARS-CoV-2-specific immunofluorescence, Western blot, in-cell ELISA, viral yield reduction and RT-qPCR methods, we demonstrated the following: (i) IMU-838 and back-ups show anti-SARS-CoV-2 activity at several levels of viral replication, i.e., protein production, double-strand RNA synthesis, and release of infectious virus; (ii) antiviral efficacy in Vero cells was demonstrated in a micromolar range (IMU-838 half-maximal effective concentration, EC of 7.6 ± 5.8 µM); (iii) anti-SARS-CoV-2 activity was distinct from cytotoxic effects (half-cytotoxic concentration, CC >100 µM); (iv) the drug in vitro potency was confirmed using several Vero lineages and human cells; (v) combination with remdesivir showed enhanced anti-SARS-CoV-2 activity; (vi) vidofludimus, the active determinant of IMU-838, exerted a broad-spectrum activity against a selection of major human pathogenic viruses. These findings strongly suggest that developmental DHODH inhibitors represent promising candidates for use as anti-SARS-CoV-2 therapeutics.
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http://dx.doi.org/10.3390/v12121394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762174PMC
December 2020

Validation of a SARS-CoV-2 RNA RT-PCR assay for high-throughput testing in blood of COVID-19 convalescent plasma donors and patients.

Transfusion 2021 02 9;61(2):368-374. Epub 2020 Nov 9.

Department of Transfusion Medicine and Hemostaseology, University Hospital of Erlangen, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany.

Background: The frequency of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNAemia in blood donors is uncertain. Thus, assays for SARS-CoV-2 RNA detection in blood, validated on commercially available polymerase chain reaction (PCR) systems, are required to allow a good comparability of data.

Study Design And Methods: The cobas SARS-CoV-2 dual-target reverse transcriptase PCR (RT-PCR) assay, licensed for respiratory swab SARS-CoV-2 RNA testing, was validated for detection of viral RNA in blood. For the validation panel, SARS-CoV-2-positive plasma samples were prepared by spiking SARS-CoV-2-positive respiratory specimens in negative human plasma. The 95% limit of detection (LOD95) was determined by probit analysis. For clinical validation, coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) donors and patients with COVID-19 with a severe disease course treated in an intensive care unit (ICU) were included.

Results: The validation of the SARS-CoV-2 RT-PCR assay for blood demonstrated high sensitivity and specificity and intra- and inter-assay precision and efficiency. The LOD95 for SARS-CoV-2 RNA was 5.0 genome copies/mL (95% confidence interval [CI], 3.3-12 copies/mL) for target 1 and 4.3 genome copies/mL (95% CI, 2.9-10 copies/mL) for target 2. In a cohort of 39 CCP donors with 66 CCP donations no SARS-CoV-2 RNA in plasma was detected. Screening of 25 blood samples of 19 ICU patients with COVID-19 showed six positive results for SARS-CoV-2 RNA in at least one target of the assay.

Conclusion: The SARS-CoV-2 RNA assay, only licensed for respiratory swabs, performed on a PCR system for high-throughput testing, showed a good assay performance for blood testing.
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http://dx.doi.org/10.1111/trf.16178DOI Listing
February 2021

Conjugation of Native-Like HIV-1 Envelope Trimers onto Liposomes Using EDC/Sulfo-NHS Chemistry: Requirements and Limitations.

Pharmaceutics 2020 Oct 16;12(10). Epub 2020 Oct 16.

Department of Biotechnology, University of Natural Resources and Life Sciences, 1190 Vienna, Austria.

The display of native-like human immunodeficiency virus type 1 envelope (HIV-1 Env) trimers on liposomes has gained wide attention over the last few years. Currently, available methods have enabled the preparation of Env-liposome conjugates of unprecedented quality. However, these protocols require the Env trimer to be tagged and/or to carry a specific functional group. For this reason, we have investigated -(3-Dimethylaminopropyl)-'-ethylcarbodiimide/-Hydroxysulfosuccinimide (EDC/Sulfo-NHS) chemistry for its potential to covalently conjugate tag-free, non-functionalized native-like Env trimers onto the surface of carboxyl-functionalized liposomes. The preservation of the liposome's physical integrity and the immunogen's conformation required a fine-tuned two-step approach based on the controlled use of β-mercaptoethanol. The display of Env trimers was strictly limited to activated liposomes of positive charge, i.e., liposomes with a positive zeta potential that carry amine-reactive Sulfo-NHS esters on their surface. In agreement with that, conjugation was found to be highly ionic strength- and pH-dependent. Overall, we have identified electrostatic pre-concentration (i.e., close proximity between negatively charged Env trimers and positively charged liposomes established through electrostatic attraction) to be crucial for conjugation reactions to proceed. The present study highlights the requirements and limitations of potentially scalable EDC/Sulfo-NHS-based approaches and represents a solid basis for further research into the controlled conjugation of tag-free, non-functionalized native-like Env trimers on the surface of liposomes, and other nanoparticles.
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http://dx.doi.org/10.3390/pharmaceutics12100979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589475PMC
October 2020

Rapid response flow cytometric assay for the detection of antibody responses to SARS-CoV-2.

Eur J Clin Microbiol Infect Dis 2021 Apr 20;40(4):751-759. Epub 2020 Oct 20.

Institute of Clinical and Molecular Virology, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany.

SARS-CoV-2 has emerged as a previously unknown zoonotic coronavirus that spread worldwide causing a serious pandemic. While reliable nucleic acid-based diagnostic assays were rapidly available, only a limited number of validated serological assays were available in the early phase of the pandemic. Here, we evaluated a novel flow cytometric approach to assess spike-specific antibody responses.HEK 293T cells expressing SARS-CoV-2 spike protein in its natural confirmation on the surface were used to detect specific IgG and IgM antibody responses in patient sera by flow cytometry. A soluble angiotensin-converting-enzyme 2 (ACE-2) variant was developed as external standard to quantify spike-specific antibody responses on different assay platforms. Analyses of 201 pre-COVID-19 sera proved a high assay specificity in comparison to commercially available CLIA and ELISA systems, while also revealing the highest sensitivity in specimens from PCR-confirmed SARS-CoV-2-infected patients. The external standard allowed robust quantification of antibody responses among different assay platforms. In conclusion, our newly established flow cytometric assay allows sensitive and quantitative detection of SARS-CoV-2-specific antibodies, which can be easily adopted in different laboratories and does not rely on external supply of assay kits. The flow cytometric assay also provides a blueprint for rapid development of serological tests to other emerging viral infections.
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http://dx.doi.org/10.1007/s10096-020-04072-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572153PMC
April 2021

CD4 T Cells Induced by Tuberculosis Subunit Vaccine H1 Can Improve the HIV-1 Env Humoral Response by Intrastructural Help.

Vaccines (Basel) 2020 Oct 13;8(4). Epub 2020 Oct 13.

Institute of Clinical and Molecular Virology, Friedrich-Alexander-University Erlangen-Nürnberg, 91054 Erlangen, Germany.

The induction of a potent and long-lasting, broadly neutralizing antibody response is one of the most promising approaches in HIV-1 vaccination. Recently, we demonstrated that Gag-specific T helper cells induced by DNA priming can enhance and modulate the HIV Env-specific B cell response upon virus-like particle (VLP) boost by intrastructural help (ISH). In order to minimize the induction of potentially harmful HIV specific T cells, we explored the possibility to harness the heterologous T cells induced by a recombinant tuberculosis subunit vaccine H1, which contains a fusion protein of Ag85B and ESAT-6 antigens in combination with the liposomal adjuvant CAF01. To provide ISH, immunodominant MHC-II restricted peptides from the H1 vaccine were genetically incorporated into the HIV 1 Gag protein and used for HIV VLP production. ISH effects on Env-specific antibody levels and B cell differentiation were analyzed in mice primed against H1 and boosted with VLPs. In contrast to non-primed mice, a significant increase of Env-specific IgG levels for up to 26 weeks after the last immunization was observed. This increase was largely caused by elevated IgG2b and IgG2c levels in mice that received H1 priming. Additionally, ISH enhanced the frequency of Env-specific long-lived plasma cells in the bone marrow. In this study, we were able to demonstrate that a heterologous prime-boost regimen consisting of the H1 tuberculosis subunit vaccine and T helper epitope modified HIV-1 VLPs resulted in enhanced HIV Env antibody and B cell responses, mediated by intrastructural help.
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http://dx.doi.org/10.3390/vaccines8040604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7711721PMC
October 2020

Genetic Co-Administration of Soluble PD-1 Ectodomains Modifies Immune Responses against Influenza A Virus Induced by DNA Vaccination.

Vaccines (Basel) 2020 Oct 1;8(4). Epub 2020 Oct 1.

Institute of Clinical and Molecular Virology, Friedrich-Alexander-University Erlangen-Nürnberg, 91054 Erlangen, Germany.

Due to the low efficacy and the need for seasonal adaptation of currently licensed influenza A vaccines, the importance of alternative vaccination strategies is increasingly recognized. Considering that DNA vaccines can be rapidly manufactured and readily adapted with novel antigen sequences, genetic vaccination is a promising immunization platform. However, the applicability of different genetic adjuvants to this approach still represents a complex challenge. Immune checkpoints are a class of molecules involved in adaptive immune responses and germinal center reactions. In this study, we immunized mice by intramuscular electroporation with a DNA-vaccine encoding hemagglutinin (HA) and nucleoprotein (NP) of the influenza A virus. The DNA-vaccine was applied either alone or in combination with genetic adjuvants encoding the soluble ectodomains of programmed cell death protein-1 (sPD-1) or its ligand (sPD-L1). Co-administration of genetic checkpoint adjuvants did not significantly alter immune responses against NP. In contrast, sPD-1 co-electroporation elevated HA-specific CD4 T cell responses, decreased regulatory CD4 T cell pools, and modulated the IgG2a-biased HA antibody pattern towards an isotype-balanced IgG response with a trend to higher influenza neutralization in vitro. Taken together, our data demonstrate that a genetic DNA-adjuvant encoding soluble ectodomains of sPD-1 was able to modulate immune responses induced by a co-administered influenza DNA vaccine.
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http://dx.doi.org/10.3390/vaccines8040570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7712647PMC
October 2020

Patients with immune-mediated inflammatory diseases receiving cytokine inhibitors have low prevalence of SARS-CoV-2 seroconversion.

Nat Commun 2020 07 24;11(1):3774. Epub 2020 Jul 24.

Department of Internal Medicine 3, Friedrich-Alexander University (FAU) Erlangen-Nuremberg and Universitätsklinikum Erlangen, Ulmenweg 18, 91054, Erlangen, Germany.

Immune-mediated inflammatory diseases (IMIDs) of the joints, gut and skin are treated with inhibitors of inflammatory cytokines. These cytokines are involved in the pathogenesis of coronavirus disease 2019 (COVID-19). Investigating anti-SARS-CoV-2 antibody responses in IMIDs we observe a reduced incidence of SARS-CoV-2 seroconversion in IMID patients treated with cytokine inhibitors compared to patients receiving no such inhibitors and two healthy control populations, despite similar social exposure. Hence, cytokine inhibitors seem to at least partially protect from SARS-CoV-2 infection.
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http://dx.doi.org/10.1038/s41467-020-17703-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382482PMC
July 2020

Targeting zonulin and intestinal epithelial barrier function to prevent onset of arthritis.

Nat Commun 2020 04 24;11(1):1995. Epub 2020 Apr 24.

Department of Internal Medicine 3, Rheumatology and Immunology, Friedrich-Alexander-University Erlangen-Nürnberg (FAU) and Universitätsklinikum Erlangen, Erlangen, Germany.

Gut microbial dysbiosis is associated with the development of autoimmune disease, but the mechanisms by which microbial dysbiosis affects the transition from asymptomatic autoimmunity to inflammatory disease are incompletely characterized. Here, we identify intestinal barrier integrity as an important checkpoint in translating autoimmunity to inflammation. Zonulin family peptide (zonulin), a potent regulator for intestinal tight junctions, is highly expressed in autoimmune mice and humans and can be used to predict transition from autoimmunity to inflammatory arthritis. Increased serum zonulin levels are accompanied by a leaky intestinal barrier, dysbiosis and inflammation. Restoration of the intestinal barrier in the pre-phase of arthritis using butyrate or a cannabinoid type 1 receptor agonist inhibits the development of arthritis. Moreover, treatment with the zonulin antagonist larazotide acetate, which specifically increases intestinal barrier integrity, effectively reduces arthritis onset. These data identify a preventive approach for the onset of autoimmune disease by specifically targeting impaired intestinal barrier function.
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http://dx.doi.org/10.1038/s41467-020-15831-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181728PMC
April 2020

Early Mortality of Brain Cancer Patients and its Connection to Cytomegalovirus Reactivation During Radiochemotherapy.

Clin Cancer Res 2020 07 14;26(13):3259-3270. Epub 2020 Feb 14.

Department of Radiation Oncology, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

Purpose: If routine diagnostics are inconclusive, neurologic deterioration and death of patients with brain cancer are attributed to tumor or therapy. Therefore, diagnosing symptoms of encephalopathy caused by human cytomegalovirus (HCMV) reactivation remains uncommon. We investigated the role of HCMV reactivation in neurologic decline and clinical outcome after the start of radiochemotherapy.

Experimental Design: HCMV analyses and extended MRI studies including additional independent retrospective neuroradiologic evaluation were performed at predetermined intervals and in case of sudden neurologic decline for 118 adult patients: 63 histologically proven high-grade gliomas, 55 with brain metastases. Immunophenotyping from simultaneously taken whole-blood samples was carried out to detect immune cells serving as prognostic marker for HCMV-associated complications. Symptomatic viremia and overall survival (OS) were the endpoints.

Results: Twenty-four percent (28/118) of all patients (12/44 glioblastoma, 3/13 anaplastic astrocytoma; 8/31 non-small cell lung cancer (NSCLC), 13/24 other brain metastases) developed HCMV-viremia during or within 4 weeks after radiotherapy; 21 of 28 patients experienced concurrent major neurologic decline, reversible by antiviral treatment. Identified by immunophenotyping, pretherapeutically low basophil counts predicted a high-risk for HCMV-associated encephalopathy (glioblastoma: = 0.002, NSCLC: = 0.02). Median OS was substantially reduced after HCMV-associated encephalopathy without MRI signs of tumor progression [glioblastoma: 99 vs. 570 days (calculated 1-year OS: 22% vs. 69%; = 0.01) and NSCLC: 47 vs. 219 days (calculated 1-year OS: 0% vs. 32%; = 0.02)].

Conclusions: For patients with brain cancer, HCMV reactivation after the start of radiochemotherapy is a frequent risk for cognitively detrimental but treatable encephalopathy and premature death. Routinely performed HCMV diagnostics, assessing basophil counts and study-based anti-viral regimens, are necessary to combat this hidden threat..
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3195DOI Listing
July 2020

Modulation of Vaccine-Induced HIV-1-Specific Immune Responses by Co-Electroporation of PD-L1 Encoding DNA.

Vaccines (Basel) 2020 Jan 14;8(1). Epub 2020 Jan 14.

Institute of Clinical and Molecular Virology, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen 91054, Germany.

The importance of a balanced T1/T2 humoral immune response against the HIV-1 envelope protein (Env) for antibody-mediated HIV-1 control is increasingly recognized. However, there is no defined vaccination strategy to raise it. Since immune checkpoints are involved in the induction of adoptive immunity and their inhibitors (monoclonal antibodies) are licensed for cancer therapy, we investigated the effect of checkpoint blockade after HIV-1 genetic vaccination on enhancement and modulation of antiviral antibody responses. By intraperitoneal administration of checkpoint antibodies in mice we observed an induction of anti-drug antibodies which may interfere with immunomodulation by checkpoint inhibitors. Therefore, we blocked immune checkpoints locally by co-electroporation of DNA vaccines encoding the active soluble ectodomains of programmed cell death protein-1 (PD-1) or its ligand (PD-L1), respectively. Plasmid-encoded immune checkpoints did not elicit a detectable antibody response, suggesting no interference with their immunomodulatory effects. Co-electroporation of a HIV-1 DNA vaccine formulation with soluble PD-L1 ectodomain increased HIV-1 Env-specific T1 CD4 T cell and IgG2a antibody responses. The overall antibody response was hereby shifted towards a more T1/T2 balanced subtype pattern. These findings indicate that co-electroporation of soluble checkpoint ectodomains together with DNA-based vaccines has modulatory effects on vaccine-induced immune responses that could improve vaccine efficacies.
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http://dx.doi.org/10.3390/vaccines8010027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157229PMC
January 2020

Characterization of a universal screening approach for congenital CMV infection based on a highly-sensitive, quantitative, multiplex real-time PCR assay.

PLoS One 2020 9;15(1):e0227143. Epub 2020 Jan 9.

Institute of Clinical and Molecular Virology, University Hospital Erlangen, Erlangen, Germany.

The majority of congenital cytomegalovirus (cCMV) infections are asymptomatic at birth and therefore not diagnosed. Approximately 10-15% of these infants develop late-onset hearing loss and other developmental disorders. Implementation of a universal screening approach at birth may allow early initiation of symptomatic interventions due to a closer follow-up of infants at risk and offers the opportunity to consider treatment of late-onset disease. Real-time PCR assays for the detection of CMV DNA in buccal swab samples demonstrated feasibility and good clinical sensitivity in comparison to a rapid culture screening assay. Because most cCMV infections remain asymptomatic, a universal screening assay that stratifies CMV infected infants according to low and high risk of late-onset cCMV disease could limit the parental anxiety and reduce follow-up costs. We therefore developed and characterized a screening algorithm based on a highly-sensitive quantitative real-time PCR assay that is compatible with centralized testing of samples from universal screening and allows to determine CMV DNA load of saliva samples either as International Units (IU)/ml saliva or IU/105 cell equivalents. 18 of 34 saliva samples of newborns that tested positively by the screening algorithm were confirmed by detection of CMV DNA in blood and/or urine samples obtained during the first weeks of life. All screening samples that could not be confirmed had viral loads of <2.3x105 IU/ml saliva (median: 6.8x103) or 1.3x105 IU/105 cell equivalents (median: 4.0x102). The viral load of screening samples with confirmed cCMV infection ranged from 7.5x102 to 8.2x109 IU/ml saliva (median: 9.3x107) or 1.5x102 to 5.6x1010 IU/105 cell equivalents (median: 3.5x106). Clinical follow-up of these newborns with confirmed cCMV infection should reveal whether the risk of late-onset cCMV disease correlates with CMV DNA load in early life saliva samples and whether a cut-off can be defined identifying cCMV infected infants with or without risk for late-onset cCMV disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0227143PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952102PMC
May 2020

Electrostatically Driven Encapsulation of Hydrophilic, Non-Conformational Peptide Epitopes into Liposomes.

Pharmaceutics 2019 Nov 18;11(11). Epub 2019 Nov 18.

Department of Biotechnology, University of Natural Resources and Life Sciences, 1190 Vienna, Austria.

Since the first use of liposomes as carriers for antigens, much work has been done to elucidate the mechanisms involved in the encapsulation of vaccine-relevant biomolecules. However, only a few studies have specifically investigated the encapsulation of hydrophilic, non-conformational peptide epitopes. We performed comprehensive and systematic screening studies, in order to identify conditions that favor the electrostatic interaction of such peptides with lipid membranes. Moreover, we have explored bi-terminal sequence extension as an approach to modify the isoelectric point of peptides, in order to modulate their membrane binding behavior and eventually shift/expand the working range under which they can be efficiently encapsulated in an electrostatically driven manner. The findings of our membrane interaction studies were then applied to preparing peptide-loaded liposomes. Our results show that the magnitude of membrane binding observed in our exploratory in situ setup translates to corresponding levels of encapsulation efficiency in both of the two most commonly employed methods for the preparation of liposomes, i.e., thin-film hydration and microfluidic mixing. We believe that the methods and findings described in the present studies will be of use to a wide audience and can be applied to address the ongoing relevant issue of the efficient encapsulation of hydrophilic biomolecules.
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http://dx.doi.org/10.3390/pharmaceutics11110619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920922PMC
November 2019

Calcium Phosphate Nanoparticle-Based Vaccines as a Platform for Improvement of HIV-1 Env Antibody Responses by Intrastructural Help.

Nanomaterials (Basel) 2019 Sep 27;9(10). Epub 2019 Sep 27.

Institute of Clinical and Molecular Virology, Friedrich-Alexander University Erlangen-Nürnberg, 91054 Erlangen, Germany.

Incorporation of immunodominant T-helper epitopes of licensed vaccines into virus-like particles (VLP) allows to harness T-helper cells induced by the licensed vaccines to provide intrastructural help (ISH) for B-cell responses against the surface proteins of the VLPs. To explore whether ISH could also improve antibody responses to calcium phosphate (CaP) nanoparticle vaccines we loaded the nanoparticle core with a universal T-helper epitope of Tetanus toxoid (p30) and functionalized the surface of CaP nanoparticles with stabilized trimers of the HIV-1 envelope (Env) resulting in Env-CaP-p30 nanoparticles. In contrast to soluble Env trimers, Env containing CaP nanoparticles induced activation of naïve Env-specific B-cells in vitro. Mice previously vaccinated against Tetanus raised stronger humoral immune responses against Env after immunization with Env-CaP-p30 than mice not vaccinated against Tetanus. The enhancing effect of ISH on anti-Env antibody levels was not attended with increased Env-specific IFN-γ CD4 T-cell responses that otherwise may potentially influence the susceptibility to HIV-1 infection. Thus, CaP nanoparticles functionalized with stabilized HIV-1 Env trimers and heterologous T-helper epitopes are able to recruit heterologous T-helper cells induced by a licensed vaccine and improve anti-Env antibody responses by intrastructural help.
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http://dx.doi.org/10.3390/nano9101389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6835376PMC
September 2019

Glycosylation of HIV Env Impacts IgG Subtype Responses to Vaccination.

Viruses 2019 02 13;11(2). Epub 2019 Feb 13.

Institute of Clinical and Molecular Virology, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nürnberg, Germany; Medical Immunology Campus Erlangen, FAU Erlangen-Nürnberg, 91054 Erlangen, Germany.

The envelope protein (Env) is the only surface protein of the human immunodeficiency virus (HIV) and as such the exclusive target for protective antibody responses. Experimental evidences from mouse models suggest a modulating property of Env to steer antibody class switching towards the less effective antibody subclass IgG1 accompanied with strong TH2 helper responses. By simple physical linkage we were able to imprint this bias, exemplified by a low IgG2a/IgG1 ratio of antigen-specific antibodies, onto an unrelated antigen, namely the HIV capsid protein p24. Here, our results indicate the glycan moiety of Env as the responsible immune modulating activity. Firstly, in mice lacking specific C-Type lectin responsiveness, DNA immunization significantly increased the IgG2a/IgG1 ratio for the Env-specific antibodies while the antibody response against the F-protein of the respiratory syncytial virus (RSV) serving as control antigen remained unchanged. Secondly, sequential shortening of the Env encoding sequence revealed the C2V3 domain as responsible for the strong IgG1 responses and TH2 cytokine production. Removing all potential N-glycosylation sites from the C2V3 domain by site-specific mutagenesis reversed the vaccine-induced immune response towards a Th1-dominated T-cell response and a balanced IgG2a/IgG1 ratio. Accordingly, the stretch of oligomannose glycans in the C2V3 domain of Env might mediate a specific uptake and/or signaling modus in antigen presenting cells by involving interaction with an as yet unknown C-type lectin receptor. Our results contribute to a deeper understanding of the impact of Env glycosylation on HIV antigen-specific immune responses, which will further support HIV vaccine development.
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http://dx.doi.org/10.3390/v11020153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6410111PMC
February 2019

Intrastructural Help: Harnessing T Helper Cells Induced by Licensed Vaccines for Improvement of HIV Env Antibody Responses to Virus-Like Particle Vaccines.

J Virol 2018 07 29;92(14). Epub 2018 Jun 29.

Institute of Clinical and Molecular Virology, University Hospital Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany.

Induction of persistent antibody responses by vaccination is generally thought to depend on efficient help by T follicular helper cells. Since the T helper cell response to HIV Env may not be optimal, we explored the possibility of improving the HIV Env antibody response to virus-like particle (VLP) vaccines by recruiting T helper cells induced by commonly used licensed vaccines to provide help for Env-specific B cells. B cells specific for the surface protein of a VLP can internalize the entire VLP and thus present peptides derived from the surface and core proteins on their major histocompatibility complex class II (MHC-II) molecules. This allows T helper cells specific for the core protein to provide intrastructural help for B cells recognizing the surface protein. Consistently, priming mice with an adjuvanted Gag protein vaccine enhanced the HIV Env antibody response to subsequent booster immunizations with HIV VLPs. To harness T helper cells induced by the licensed Tetanolpur vaccines, HIV VLPs that contained T helper cell epitopes of tetanus toxoid were generated. Tetanol-immunized mice raised stronger antibody responses to immunizations with VLPs containing tetanus toxoid T helper cell epitopes but not to VLPs lacking these epitopes. Depending on the priming immunization, the IgG subtype response to HIV Env after the VLP immunization could also be modified. Thus, harnessing T helper cells induced by other vaccines appears to be a promising approach to improve the HIV Env antibody response to VLP vaccines. Induction of HIV Env antibodies at sufficient levels with optimal Fc effector functions for durable protection remains a challenge. Efficient T cell help may be essential to induce such a desirable antibody response. Here, we provide proof of concept that T helper cells induced by a licensed vaccine can be harnessed to provide help for HIV Env-specific B cells and to modulate the Env-specific IgG subtype response.
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http://dx.doi.org/10.1128/JVI.00141-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6026751PMC
July 2018

Dendritic cell targeted HIV-1 gag protein vaccine provides help to a recombinant Newcastle disease virus vectored vaccine including mobilization of protective CD8 T cells.

Immun Inflamm Dis 2018 03 4;6(1):163-175. Epub 2017 Dec 4.

Laboratory of Vaccinology/Biobanking of The Chantal Biya International Reference Center for research on the prevention and management of HIV/AIDS (CIRCB), BP 3077, Messa Yaounde, Cameroon.

Introduction: Recombinant Newcastle Disease virus (rNDV) vectored vaccines are safe mucosal applicable vaccines with intrinsic immune-modulatory properties for the induction of efficient immunity. Like all viral vectored vaccines repeated inoculation via mucosal routes invariably results to immunity against viral vaccine vectors. To obviate immunity against viral vaccine vectors and improve the ability of rNDV vectored vaccines in inducing T cell immunity in murine air way we have directed dendritic cell targeted HIV-1 gag protein (DEC-Gag) vaccine; for the induction of helper CD4 T cells to a Recombinant Newcastle disease virus expressing codon optimized HIV-1 Gag P55 (rNDV-L-Gag) vaccine.

Methods: We do so through successive administration of anti-DEC205-gagP24 protein plus polyICLC (DEC-Gag) vaccine and rNDV-L-Gag. First strong gag specific helper CD4 T cells are induced in mice by selected targeting of anti-DEC205-gagP24 protein vaccine to dendritic cells (DC) in situ together with polyICLC as adjuvant. This targeting helped T cell immunity develop to a subsequent rNDV-L-Gag vaccine and improved both systemic and mucosal gag specific immunity.

Results: This sequential DEC-Gag vaccine prime followed by an rNDV-L-gag boost results to improved viral vectored immunization in murine airway, including mobilization of protective CD8 T cells to a pathogenic virus infection site.

Conclusion: Thus, complementary prime boost vaccination, in which prime and boost favor distinct types of T cell immunity, improves viral vectored immunization, including mobilization of protective CD8 T cells to a pathogenic virus infection site such as the murine airway.
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http://dx.doi.org/10.1002/iid3.209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818444PMC
March 2018

[Attenuated zoster vaccine is not recommended as a standard vaccine : Missed opportunity or logical implementation of the evidence-based approach of the Standing Committee on Vaccination].

Authors:
Klaus Überla

Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 2017 Oct;60(10):1065-1066

Virologisches Institut, Klinische und Molekulare Virologie, Universitätsklinikum Erlangen, Schlossgarten 4, 91054, Erlangen, Deutschland.

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http://dx.doi.org/10.1007/s00103-017-2632-8DOI Listing
October 2017

Vector Order Determines Protection against Pathogenic Simian Immunodeficiency Virus Infection in a Triple-Component Vaccine by Balancing CD4 and CD8 T-Cell Responses.

J Virol 2017 12 14;91(23). Epub 2017 Nov 14.

Unit of Infection Models, Deutsches Primatenzentrum GmbH, Goettingen, Germany

An effective AIDS vaccine should elicit strong humoral and cellular immune responses while maintaining low levels of CD4 T-cell activation to avoid the generation of target cells for viral infection. The present study investigated two prime-boost regimens, both starting vaccination with single-cycle immunodeficiency virus, followed by two mucosal boosts with either recombinant adenovirus (rAd) or fowlpox virus (rFWPV) expressing SIVmac239 or SIVmac251 and genes, respectively. Finally, vectors were switched and systemically administered to the reciprocal group of animals. Only mucosal rFWPV immunizations followed by systemic rAd boost significantly protected animals against a repeated low-dose intrarectal challenge with pathogenic SIVmac251, resulting in a vaccine efficacy (i.e., risk reduction per exposure) of 68%. Delayed viral acquisition was associated with higher levels of activated CD8 T cells and Gag-specific gamma interferon (IFN-γ)-secreting CD8 cells, low virus-specific CD4 T-cell responses, and low Env antibody titers. In contrast, the systemic rFWPV boost induced strong virus-specific CD4 T-cell activity. rAd and rFWPV also induced differential patterns of the innate immune responses, thereby possibly shaping the specific immunity. Plasma levels after final immunization correlated directly with virus-specific CD4 T-cell responses and inversely with the number of exposures to infection. Also, the percentage of activated CD69 CD8 T cells correlated with the number of exposures to infection. Differential stimulation of the immune response likely provided the basis for the diverging levels of protection afforded by the vaccine regimen. A failed phase II AIDS vaccine trial led to the hypothesis that CD4 T-cell activation can abrogate any potentially protective effects delivered by vaccination or promote acquisition of the virus because CD4 T helper cells, required for an effective immune response, also represent the target cells for viral infection. We compared two vaccination protocols that elicited similar levels of Gag-specific immune responses in rhesus macaques. Only the animal group that had a low level of virus-specific CD4 T cells in combination with high levels of activated CD8 T cells was significantly protected from infection. Notably, protection was achieved despite the lack of appreciable Env antibody titers. Moreover, we show that both the vector and the route of immunization affected the level of CD4 T-cell responses. Thus, mucosal immunization with FWPV-based vaccines should be considered a potent prime in prime-boost vaccination protocols.
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http://dx.doi.org/10.1128/JVI.01120-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5686736PMC
December 2017

Generation of T follicular helper cells in vitro: requirement for B-cell receptor cross-linking and cognate B- and T-cell interaction.

Immunology 2018 02 9;153(2):214-224. Epub 2017 Oct 9.

Institute of Clinical and Molecular Virology, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany.

The minimum requirements for in vitro modelling of natural CD4 T-cell differentiation into T follicular helper (Tfh) cells are still under investigation. We co-cultured wild-type and T-cell receptor (TCR) transgenic CD4 T cells from naive mice with dendritic cells and B-cell receptor (BCR) transgenic B cells in the presence of HIV-derived virus-like particles containing matched B-cell and T-cell epitopes. This co-culturing induced co-expression of Tfh-master regulator transcription factor BCL-6 and CXCR5 in up to 10% of the wild-type and up to 40% of the TCR-transgenic CD4 T cells. Phenotypic markers, production of interleukin-21 and isotype switching of the B cells to IgG1 further indicated a helper function of the induced Tfh cells in vitro. Dendritic cells supported the generation of functional Tfh cells, but were unable to induce them without cognate B cells. Hence, our study presents a robust experimental system for efficient generation of functionally active Tfh cells in vitro and confirms the importance of cognate B- and T-cell cross-talk for the Tfh differentiation process.
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http://dx.doi.org/10.1111/imm.12834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765376PMC
February 2018

A clinician's plea to test glioma patients for CMV.

Neuro Oncol 2017 09;19(9):1282-1283

Department of Radiation Oncology, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany; Institute of Clinical and Molecular Virology, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

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http://dx.doi.org/10.1093/neuonc/nox080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570148PMC
September 2017

In vivo targeting of protein antigens to dendritic cells using anti-DEC-205 single chain antibody improves HIV Gag specific CD4 T cell responses protecting from airway challenge with recombinant vaccinia-gag virus.

Immun Inflamm Dis 2019 06 13;7(2):55-67. Epub 2017 Mar 13.

Laboratory of Vaccinology/Biobanking of The Chantal Biya International Reference Center for Research on The Prevention and Management of HIV/AIDS, Yaounde, Cameroon.

Introduction: Targeting antigens to dendritic cells (DCs) in vivo via a DC-restricted endocytic receptor, DEC205, has been validated to enhance immunity in several vaccine platforms. Particularly atttractive is selected delivery of proteins to DCs in vivo because it enables proteins to be more immunogenic and provides a cheaper and effective way for repeated immunizations.

Methods: In this study, we tested the efficacy of a single chain antibody to DEC205 (scDEC) to deliver protein antigens selectively to DCs in vivo and to induce protective immunity.

Results: In comparison to soluble Ovalbumin (OVA) antigen, when recombinant scDEC:OVA protein was injected subcutaneously (s.c.) into mice, the OVA protein was selectively presented by DCs to both TCR transgenic CD8 and CD4 T cells approximately 500 and 100 times more efficient than soluble OVA, respectively, and could persist for seven days following s.c. injection of the scDEC205:OVA. Similarly selective targeting of HIV Gag P24 to DCs in vivo using scDEC-Gag protein plus polyICLC vaccine resulted in strong, long lasting, polyfuntional CD4 T cells in mice which were protective against airway challenge by a recombinant vaccinia-gag virus.

Conclusion: Thus targeting protein antigens to DCs using scDEC can be used either alone or in combination with other strategies for effective immunization.
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http://dx.doi.org/10.1002/iid3.151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485703PMC
June 2019

Intrastructural help: improving the HIV-1 envelope antibody response induced by virus-like particle vaccines.

Curr Opin HIV AIDS 2017 May;12(3):272-277

Institute of Clinical and Molecular Virology, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany.

Purpose Of Review: The importance of IgG Fc-effector functions for the efficacy of HIV vaccines is increasingly recognized. Although different types of vaccines were shown to induce antibodies with different Fc-activities, there is no clear strategy how to raise antibody responses with a desired pattern of Fc-effector functions. Given the central role of T-helper cells in regulating the germinal center reaction and the differentiation of B cells in an antigen-specific manner, the review will discuss whether T-helper cells directed against non-HIV envelope (Env) antigens could be harnessed to improve the HIV-Env antibody response.

Recent Findings: Comparing CD4 T-cell responses in HIV-infected individuals with and without neutralizing antibody breadth suggests that robust Gag-specific CD4 T cells may provide important T-cell help to Env-specific B cells. In a murine model, GagPol-specific T-helper cells were shown to provide intrastructural help for HIV-Env-specific antibody responses after immunization with a virus-like particle vaccine. GagPol-specific T-helper cells imprinted the IgG subtype ratio observed for Gag onto the HIV-Env antibody response and modulated the glycosylation pattern of the HIV Env-specific antibodies.

Summary: Intrastructural help is a promising strategy to improve overall levels and Fc-effector functions of the HIV-Env antibody response.
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http://dx.doi.org/10.1097/COH.0000000000000358DOI Listing
May 2017

Application of the PAMONO-Sensor for Quantification of Microvesicles and Determination of Nano-Particle Size Distribution.

Sensors (Basel) 2017 Jan 27;17(2). Epub 2017 Jan 27.

Leibniz Institute für Analytische Wissenschaften, ISAS e.V., Bunsen-Kirchhoff-Straße 11, 44139 Dortmund, Germany.

The PAMONO-sensor (plasmon assisted microscopy of nano-objects) demonstrated an ability to detect and quantify individual viruses and virus-like particles. However, another group of biological vesicles-microvesicles (100-1000 nm)-also attracts growing interest as biomarkers of different pathologies and needs development of novel techniques for characterization. This work shows the applicability of a PAMONO-sensor for selective detection of microvesicles in aquatic samples. The sensor permits comparison of relative concentrations of microvesicles between samples. We also study a possibility of repeated use of a sensor chip after elution of the microvesicle capturing layer. Moreover, we improve the detection features of the PAMONO-sensor. The detection process utilizes novel machine learning techniques on the sensor image data to estimate particle size distributions of nano-particles in polydisperse samples. Altogether, our findings expand analytical features and the application field of the PAMONO-sensor. They can also serve for a maturation of diagnostic tools based on the PAMONO-sensor platform.
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http://dx.doi.org/10.3390/s17020244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5336007PMC
January 2017

Nanoparticle-based B-cell targeting vaccines: Tailoring of humoral immune responses by functionalization with different TLR-ligands.

Nanomedicine 2017 01 2;13(1):173-182. Epub 2016 Sep 2.

Department of Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany; University Hospital Erlangen, Institute of Clinical and Molecular Virology, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany. Electronic address:

Induction of an appropriate type of humoral immune response during vaccination is essential for protection against viral and bacterial infections. We recently observed that biodegradable calcium phosphate (CaP) nanoparticles coated with proteins efficiently targeted and activated naïve antigen-specific B-cells in vitro. We now compared different administration routes for CaP-nanoparticles and demonstrated that intramuscular immunization with such CaP-nanoparticles induced stronger immune responses than immunization with monovalent antigen. Additional functionalization of the CaP-nanoparticles with TRL-ligands allowed modulating the IgG subtype response and the level of mucosal IgA antibodies. CpG-containing CaP-nanoparticles were as immunogenic as a virus-like particle vaccine. Functionalization of CaP-nanoparticles with T-helper cell epitopes or CpG also allowed overcoming lack of T-cell help. Thus, our results indicate that CaP-nanoparticle-based B-cell targeting vaccines functionalized with TLR-ligands can serve as a versatile platform for efficient induction and modulation of humoral immune responses in vivo.
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http://dx.doi.org/10.1016/j.nano.2016.08.028DOI Listing
January 2017

Frequent occurrence of therapeutically reversible CMV-associated encephalopathy during radiotherapy of the brain.

Neuro Oncol 2016 12 10;18(12):1664-1672. Epub 2016 Jun 10.

Department of Radiation Oncology, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany (N.L.G., B.F., P.F.R., F.P., S.S., U.S.G., R.F.); Institute of Clinical and Molecular Virology, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany (K.K., B.F., K.U.); Institute of Neuroradiology, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany (M.A.S., A.D., T.E.); Department of Neurosurgery, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany (I.E.).

Background: Neurological decline during radio(chemo)therapy of the brain is often attributed to disease progression or side effects of radiotherapy. Diagnosis of opportunistic neurotropic infections such as cytomegalovirus (CMV) infections is uncommon, even though high-grade gliomas and some brain metastases are known to contain CMV particles. We prospectively examined the frequency of CMV encephalopathy during radiotherapy of the brain.

Methods: Fifty patients requiring whole-brain radiotherapy for brain metastases (n = 27) or local radio(chemo)therapy of the brain for high-grade gliomas (n = 23) were observed in the prospective observational GLIO-CMV-01 study. MRIs and blood samples were obtained before, halfway through, and at the end of radiotherapy. MRIs were screened for disease progression or increased intracranial pressure. Blood was tested for anti-CMV immunoglobulin (Ig)M, anti-CMV IgG, and CMV DNA.

Results: Thirty-two of 50 (64%) patients were positive for anti-CMV IgG before radio(chemo)therapy. Fifteen of those 32 (48%) developed viremia during or up to 28 days after treatment. Thirteen of those 15 (87%) required treatment for CMV-associated encephalopathy. MRIs were negative for disease progression, edema, or bleeding. None of the patients negative for anti-CMV IgG developed viremia, suggesting a reactivation rather than a primary infection.In the group at risk consisting of anti-CMV IgG+ patients, age >65 (P = .004) and the amount of dexamethasone taken during radio(chemo)therapy (P = .004) were associated with an increased risk for CMV-associated encephalopathy. One hundred and fifty days after the start of radio(chemo)therapy, survival was 74% (14/19) (no encephalopathy) versus 54% (7/13) (encephalopathy) (odds ratio, 0.42; 95% CI, 0.03-1.86; P = .25).

Conclusion: CMV reactivation frequently causes encephalopathy during radio(chemo)therapy of the brain. The unexpected high incidence of this infection makes it highly clinically relevant for every treating physician.
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http://dx.doi.org/10.1093/neuonc/now120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5793811PMC
December 2016

The improved antibody response against HIV-1 after a vaccination based on intrastructural help is complemented by functional CD8+ T cell responses.

Vaccine 2016 Apr 3;34(15):1744-51. Epub 2016 Mar 3.

Department of Molecular and Medical Virology, Ruhr-University Bochum, Germany. Electronic address:

Despite more than three decades of intense research, a prophylactic HIV-1 vaccine remains elusive. Four vaccine modalities have been evaluated in clinical efficacy studies, but only one demonstrated at least modest efficacy, which correlated with polyfunctional antibody responses to the HIV surface protein Env. To be most effective, a HIV-1 vaccine probably has to induce both, functional antibody and CD8(+) T cell responses. We therefore analyzed DNA/DNA and DNA/virus-like particle (VLP) regimens for their ability to induce humoral and cellular immune responses. Here, DNA vaccination of mice induced strong CD8(+) responses against Env and Gag. However, the humoral response to Env was dominated by IgG1, a subclass known for its low functionality. In contrast, priming only with the Gag-encoding plasmid followed by a boost with VLPs consisting of Gag and Env improved the quality of the anti-Env antibody response via intrastructural help (ISH) provided by Gag-specific T cells to Env-specific B cells. Furthermore, the Gag-specific CD8(+) T cells induced by the DNA prime immunization could still protect from a lethal infection with recombinant vaccinia virus encoding HIV Gag. Therefore, this immunization regimen represents a promising approach to combine functional antibody responses toward HIV Env with strong CD8(+) responses controlling early viral replication.
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http://dx.doi.org/10.1016/j.vaccine.2016.02.059DOI Listing
April 2016