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.
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
. The objective of the current study was to use so-called photochemical internalization (PCI) to deliver a live bacterial vaccine to the cytosol of antigen-presenting cells (APCs) for the purpose of stimulating major histocompatibility complex (MHC) I-restricted CD8 T-cell responses. For this purpose,
BCG (BCG) was combined with the photosensitiser tetraphenyl chlorine disulfonate (TPCS2a) and injected intradermally into mice. TPCS2a was then activated by illumination of the injection site with light of defined energy. Antigen-specific CD4 and CD8 T-cell responses were monitored in blood, spleen, and lymph nodes at different time points thereafter using flow cytometry, ELISA and ELISPOT. Finally, APCs were infected and PCI-treated
for analysis of their activation of T cells
or
after autologous vaccination of mice. Combination of BCG with PCI induced stronger BCG-specific CD4 and CD8 T-cell responses than treatment with BCG only or with BCG and TPCS2a without light. The overall T-cell responses were multifunctional as characterized by the production of IFN-γ, TNF-α, IL-2 and IL-17. Importantly, PCI induced cross-presentation of BCG proteins for stimulation of antigen-specific CD8 T-cells that were particularly producing IFN-γ and TNF-α. PCI further facilitated antigen presentation by causing up-regulation of MHC and co-stimulatory proteins on the surface of APCs as well as their production of TNF-α and IL-1β
. Furthermore, PCI-based vaccination also caused local inflammation at the site of vaccination, showing strong infiltration of immune cells, which could contribute to the stimulation of antigen-specific immune responses. This study is the first to demonstrate that a live microbial vaccine can be combined with a photochemical compound and light for cross presentation of antigens to CD8 T cells. Moreover, the results revealed that PCI treatment strongly improved the immunogenicity of
BCG.
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-derived protocol for assessment of neutralization of SARS-CoV-2 is described. Compared with the often applied plaque-forming unit assay, the working load is lower, and fewer manipulations of the infected cultures are required. Hence, the method is safer for the personnel.
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•The protocol describes assessment of neutralization of SARS-CoV-2 by antibodies•Neutralization is detected by means of reduced cytopathic effect on adherent cells•Increased safety for researchers through less manipulation of infectious cultures•The described protocol allows for more efficient upscaling of sample testing
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-derived protocol for assessment of neutralization of SARS-CoV-2 is described. Compared with the often applied plaque-forming unit assay, the working load is lower, and fewer manipulations of the infected cultures are required. Hence, the method is safer for the personnel.
Abstract Vaccines generally require T lymphocytes for B-cell activation and immunoglobulin class switching in response to peptide or protein antigens. In the absence of T cells, limited IgG class ...switch takes place, germinal centers are short-lived, and the B cells lack memory. Here, immunization of mice with liposomes containing 15mer peptides and monophosphoryl lipid A (MPLA) as adjuvant, induced T-cell independent (TI) IgG class switch within three days, as well as germinal center formation. The antibody responses were long-lived, strictly dependent on Toll-like receptor 4 (TLR4) signaling, partly dependent on Bruton’s tyrosine kinase (BTK) signal transmission, and independent of signaling through T-cell receptors, MHC class II and inflammasome. The antibody response showed characteristics of both TI type 1 and TI type 2. All IgG subclasses could be boosted months after primary immunization, and the biological function of the secreted antibodies was demonstrated in murine models of allergic anaphylaxis and of bacterial infection. Moreover, antibody responses after immunization with peptide- and MPLA-loaded liposomes could be triggered in neonatal mice and in mice receiving immune-suppressants. This study demonstrates T-cell independent endogenous B-cell memory and recall responses in vivo using a peptide antigen. The stimulation of these antibody responses required a correct and dense assembly and administration of peptide and adjuvant on the surface of liposomes. In the future, TI vaccines may prove beneficial in pathological conditions in which T-cell immunity is compromised through disease or medicines or when rapid, antibody-mediated immune protection is needed.
Introduction
Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), substantial effort has been made to gain knowledge about the immunity elicited by infection or ...vaccination.
Methods
We studied the kinetics of antibodies and virus neutralisation induced by vaccination with BNT162b2 in a Swiss cohort of SARS‐CoV‐2 naïve (n = 40) and convalescent (n = 9) persons. Blood sera were analysed in a live virus neutralisation assay and specific IgG and IgA levels were measured by enzyme‐linked immunoassay and analysed by descriptive statistics.
Results
Virus neutralisation was detected in all individuals 2–4 weeks after the second vaccine. Both neutralisation and antibodies remained positive for >4 months. Neutralisation and antibodies showed positive correlation, but immunoglobulin G (IgG) and immunoglobulin A (IgA) seroconversion took place 2–4 weeks faster than neutralisation. Spike‐protein specific IgG levels rose significantly faster and were more stable over time than virus neutralisation titres or IgA responses. For naïve but not convalescent persons, a clear boosting effect was observed. Convalescent individuals showed faster, more robust and longer‐lasting immune responses after vaccination compared to noninfected persons. No threshold could be determined for spike protein‐specific IgG or IgA that would confer protection in the neutralisation assay, implicating the need for a better correlate of protection then antibody titres alone.
Conclusions
This study clearly shows the complex translation of antibody data and virus neutralisation, while supporting the evidence of a single dose being sufficient for effective antibody response in convalescent individuals.
The kinetics of antibodies and virus neutralisation induced by vaccination with BNT162b2 was studied. Neutralisation and antibodies remained positive for >4 months. Immunoglobulin G (IgG) and immunoglobulin A (IgA) seroconversion took place 2–4 weeks faster than neutralisation. IgG levels rose significantly faster and were more stable over time than virus neutralisation titres or IgA responses. Boosting effect was observed in naïve but not in convalescent persons. Convalescent individuals showed faster, more robust and longer‐lasting immune responses after vaccination compared to noninfected persons.
Real-time RT-PCR remains a gold standard in the detection of various viral diseases. In the coronavirus 2019 pandemic, multiple RT-PCR–based tests were developed to screen for viral infection. As an ...emergency response to increasing testing demand, we established a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PCR diagnostics platform for which we compared different commercial and in-house RT-PCR protocols. Four commercial, one customized, 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. In addition, a highly sensitive digital droplet PCR (ddPCR) method was developed, and application of RT-PCR 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% to 97.8%), and high specificities (98.7% to 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.
Background
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.
Objective
In this retrospective study, we aimed to assess the sensitivity and specificity of this method for the diagnosis of CTCL.
Materials and Methods
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.
Results
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.
Conclusion
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.
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.
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