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Conventional vaccines are very efficient in the prevention of bacterial infections caused by extracellular pathogens due to effective stimulation of pathogen-specific antibodies. In contrast, considering that intracellular surveillance by antibodies is not possible, they are typically less effective in preventing or treating infections caused by intracellular pathogens such as

For a cytopathic virus such as severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the neutralization capacity of serum from convalescent or vaccinated persons or of therapeutic antibodies can be tested on adherent cell cultures. Here, a simple and tissue culture infectious dose (TCID)-derived protocol for assessment of neutralization of SARS-CoV-2 is described. Compared to the often applied plaque-forming unit (PFU) assay, the working load is lower, and fewer manipulations of the infected cultures are required. Hence, the method is safer for the personnel., Graphical Abstract

Real-time reverse transcription polymerase chain reaction (RT-PCR) remains a gold standard in detection of various viral diseases. In the COVID-19 pandemic, multiple RT-PCR based tests were developed to screen for viral infection. As an emergency response to growing testing demand, we established a SARS-CoV-2 PCR diagnostics platform for which we compared different commercial and in-house RT-PCR protocols. Four commercial (CDC 2019-nCoV, Applied BiosystemsTM 2019-nCoV Assay Kit v1 TF-SinglePlex, 2019-nCoV Assay Kit v2 TF-MultiPlex, and EURORealTime SARS-CoV-2), one customized (Institute Pasteur), and one in-house RT-PCR protocols were evaluated with 92 SARS-CoV-2 positive and 92 SARS-CoV-2 negative samples. Furthermore, economical and practical characteristics of these protocols were compared. Additionally, a highly sensitive digital droplet PCR (ddPCR) method was developed and application of RT- and ddPCR methods on SARS-CoV-2 environmental samples was examined. Very low limits of detection (1 or 2 viral copies/μL), high sensitivities (93.6-97.8%) and specificities (98.7-100%) for the tested RT-PCR protocols were found. Furthermore, the feasibility of downscaling two of the commercial protocols, which could optimize testing capacity was demonstrated. Tested commercial and customized RT-PCR detection kits show very good and comparable sensitivity, and specificity, and the kits could be further optimized for use on SARS-CoV-2 viral samples derived from human and surface swabbed samples.

Real-time reverse transcription polymerase chain reaction (RT-PCR) remains a gold standard in detection of various viral diseases. In the COVID-19 pandemic, multiple RT-PCR based tests were developed to screen for viral infection. As an emergency response to growing testing demand, we established a SARS-CoV-2 PCR diagnostics platform for which we compared different commercial and in-house RT-PCR protocols. We evaluated four commercial (CDC 2019-nCoV, Applied Biosystems™ 2019-nCoV Assay Kit v1 TF-SinglePlex, 2019-nCoV Assay Kit v2 TF-MultiPlex, and EURORealTime SARS-CoV-2), one customized (Institute Pasteur), and one in-house RT-PCR protocols with 92 SARS-CoV-2 positive and 92 SARS-CoV-2 negative samples. Furthermore, we compared economical and practical characteristics of these protocols. We also developed a highly sensitive digital droplet PCR (ddPCR) method. Finally, we conducted a local environmental study for the presence and infectivity of SARS-CoV-2 on different surfaces in a quarantined household using RT- and ddPCR methods. We found very low limits of detection (1 or 2 viral copies/μL), high sensitivities (93.6-97.8%) and specificities (98.7-100%) for the tested RT-PCR protocols. We further demonstrated the feasibility of downscaling two of the commercial protocols, which could optimize testing capacity. In the local environmental study, only one surface sample tested positive for viral RNA, but without detectable infectivity in vitro. Tested commercial and customized RT-PCR detection kits show very good and comparable sensitivity, and specificity, and the kits could be further optimized for use on SARS-CoV-2 viral samples derived from human and surface swabbed samples.

Early and precise diagnosis of cutaneous T-cell lymphomas (CTCL) is challenging. Currently, polymerase chain reaction (PCR)-based clonality assessment of the T-cell receptor (TCR) is a helpful tool for this diagnosis.In this retrospective study, we aimed to assess the sensitivity and specificity of this method for the diagnosis of CTCL.Monoclonal rearrangement of the TCR was investigated retrospectively by PCR-based clonality assessment based on 292 DNA samples from skin biopsies of patients with a suspicion of CTCL. Algorithms were based on different ratios (three or five-fold difference) between the dominant PCR peak and the third highest PCR peak.A PCR peak five-fold higher than the third highest PCR peak demonstrated significantly greater specificity (83.7% versus 76.4%) but lower sensitivity (59.8% versus 68.6%) compared to a cut-off of three-fold higher.Our results confirm the diagnostic value of TCR clonality analysis which may be used to define the ideal cut-off in order to optimize sensitivity and specificity.

Allergen immunotherapy (AIT) is the only modality that can modify immune responses to allergen exposure, but therapeutic coverage is low. One strategy to improve AIT safety and efficacy is the use of new or improved adjuvants. This study investigates immune responses produced by microcrystalline tyrosine (MCT)–based vaccines as compared with conventional aluminum hydroxide (alum). Wild-type, immune-signaling–deficient, and TCR-transgenic mice were treated with different Ags (e.g., OVA and cat dander Fel d 1), plus MCT or alum as depot adjuvants. Specific Ab responses in serum were measured by ELISA, whereas cytokine secretion was measured both in culture supernatants by ELISA or by flow cytometry of spleen cells. Upon initiation of AIT in allergic mice, body temperature and further clinical signs were used as indicators for anaphylaxis. Overall, MCT and alum induced comparable B and T cell responses, which were independent of TLR signaling. Alum induced stronger IgE and IL-4 secretion than MCT. MCT and alum induced caspase-dependent IL-1β secretion in human monocytes in vitro, but inflammasome activation had no functional effect on inflammatory and Ab responses measured in vivo. In sensitized mice, AIT with MCT-adjuvanted allergens caused fewer anaphylactic reactions compared with alum-adjuvanted allergens. As depot adjuvants, MCT and alum are comparably effective in strength and mechanism of Ag-specific IgG induction and induction of T cell responses. The biocompatible and biodegradable MCT seems therefore a suitable alternative adjuvant to alum-based vaccines and AIT.

Allergen-specific subcutaneous (SCIT) as well as sublingual (SLIT) immunotherapy both confer long-term protection against the highly prevalent IgE-mediated allergies, such as allergic rhino-conjunctivitis and asthma. Since both SCIT and SLIT are both very laborious and time-consuming treatments, we and others have investigated whether the immunotherapy could be enhanced by so-called intralymphatic immunotherapy (ILIT), which aims at improving treatment efficacy and safety and to reduce treatment duration, thereby making allergen immunotherapy (AIT) more patient friendly. In the original studies made by us (1, 2) and by Cardell et al (3), ILIT was administered three times with four week intervals. In 2013, Witten et al. questioned the efficacy of ILIT with grass pollen, based on the results of a clinical study of their in 38 adult hay-fever patients (4). While promising immunological changes such as increased IgG4 and Treg responses were observed, they concluded that the clinical outcome was at odds with that measured by us in 165 hay fever and 20 cat-dander allergic patients as well as in Cardell's study with 28 hay-fever patients. This article is protected by copyright. All rights reserved.

Vaccines typically come with adjuvants that trigger the innate immune system in order to prepare best possible inflammatory conditions as to allow the adaptive immune system to become activated, generally for the induction of antibodies. The oldest approved and most abundant immunological adjuvants are salts of aluminium, which are also frequently used in animal models of immunisation and allergy desensitization. In rodents, the intraperitoneal administration of aluminium adjuvants is commonly performed and considered safe. In the current investigation, we show that intraperitoneal administration of aluminium adjuvants is associated with a dose-dependent hypothermic reaction within 10 min of the injection. The body temperature of mice dropped as much as 4 °C, and the clinical symptoms included apathy, hunched posture, and piloerection. The temperature normalised and other clinical manifestations disappeared within 60–80 min of the intraperitoneal aluminium injection, which caused strong infiltration of neutrophil and eosinophil granulocytes into the peritoneal cavity, a clinical manifestations typically associated with inflammasome activation. However, the observed reactions to aluminium adjuvants were independent of NALP3, caspase-1, and interleukin-1β, but dependent on histamine. Hence, aluminium adjuvants may have potential local and systemic side effects, which warrants further investigations into the nature of these side effects, but also into the possible implications on health in man.

Studying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO Lec3.2.8.1 cells. Using preparative cell sorting, stable, high-expressing GFP 'master' cell lines were generated that can be converted fast and reliably by targeted integration via Flp recombinase-mediated cassette exchange (RMCE) to produce any glycoprotein. Small-scale transient transfection of HEK293 cells was used to identify genetically engineered constructs suitable for constructing stable cell lines. Stable cell lines expressing 10 different proteins were established. The system was validated by expression, purification, deglycosylation and crystallization of the heavily glycosylated luminal domains of lysosome-associated membrane proteins (LAMP). © 2011 Wilke et al.