91 research outputs found
Recombinant Envelope-Proteins with Mutations in the Conserved Fusion Loop Allow Specific Serological Diagnosis of Dengue-Infections
Dengue virus (DENV) is a mosquito-borne flavivirus and a major international public health concern in many tropical and sub-tropical areas worldwide. DENV is divided into four major serotypes, and infection with one serotype leads to immunity against the same, but not the other serotypes. The specific diagnosis of DENV-infections via antibody-detection is problematic due to the high degree of cross-reactivity displayed by antibodies against related flaviviruses, such as West Nile virus (WNV), Yellow Fever virus (YFV) or Tick-borne encephalitis virus (TBEV). Especially in areas where several flaviviruses co-circulate or in the context of vaccination e.g. against YFV or TBEV, this severely complicates diagnosis and surveillance. Most flavivirus cross-reactive antibodies are produced against the highly conserved fusion loop (FL) domain in the viral envelope (E) protein. We generated insect-cell derived recombinant E-proteins of the four DENV-serotypes which contain point mutations in the FL domain. By using specific mixtures of these mutant antigens, cross-reactivity against heterologous flaviviruses was strongly reduced, enabling sensitive and specific diagnosis of the DENV-infected serum samples in IgG and IgM-measurements. These results have indications for the development of serological DENV-tests with improved specificity
Direct membrane proteinâDNA interactions required early in nuclear envelope assembly
Among the earliest events in postmitotic nuclear envelope (NE) assembly are the interactions between chromatin and the membranes that will fuse to form the NE
Uptake and fecal excretion of Coxiella burnetii by Ixodes ricinus and Dermacentor marginatus ticks
Background:
The bacterium Coxiella burnetii is the etiological agent of Q fever and is mainly transmitted via inhalation of infectious aerosols. DNA of C. burnetii is frequently detected in ticks, but the role of ticks as vectors in the epidemiology of this agent is still controversial. In this study, Ixodes ricinus and Dermacentor marginatus adults as well as I. ricinus nymphs were fed on blood spiked with C. burnetii in order to study the fate of the bacterium within putative tick vectors.
Methods:
Blood-feeding experiments were performed in vitro in silicone-membrane based feeding units. The uptake, fecal excretion and transstadial transmission of C. burnetii was examined by quantitative real-time PCR as well as cultivation of feces and crushed tick filtrates in L-929 mouse fibroblast cells and cell-free culture medium.
Results:
Ticks successfully fed in the feeding system with engorgement rates ranging from 29% (D. marginatus) to 64% (I. ricinus adults). Coxiella burnetii DNA was detected in the feces of both tick species during and after feeding on blood containing 105 or 106 genomic equivalents per ml blood (GE/ml), but not when fed on blood containing only 104 GE/ml. Isolation and cultivation demonstrated the infectivity of C. burnetii in shed feces. In 25% of the I. ricinus nymphs feeding on inoculated blood, a transstadial transmission to the adult stage was detected. Females that molted from nymphs fed on inoculated blood excreted C. burnetii of up to 106 genomic equivalents per mg of feces.
Conclusions:
These findings show that transstadial transmission of C. burnetii occurs in I. ricinus and confirm that I. ricinus is a potential vector for Q fever. Transmission from both tick species might occur by inhalation of feces containing high amounts of viable C. burnetii rather than via tick bites
T Cell epitope mapping of the E-protein of West Nile virus in BALB/c mice
West Nile virus (WNV) is a zoonotic virus, which is transmitted by mosquitoes. It is the causative agent of the disease syndrome called West Nile fever. In some human cases, a WNV infection can be associated with severe neurological symptoms. The immune response to WNV is multifactorial and includes both humoral and cellular immunity. T-cell epitope mapping of the WNV envelope (E) protein has been performed in C57BL/6 mice, but not in BALB/c mice. Therefore, we performed in BALB/c mice a T-cell epitope mapping using a series of peptides spanning the WNV envelope (E) protein. To this end, the WNV-E specific T cell repertoire was first expanded by vaccinating BALB/c mice with a DNA vaccine that generates subviral particles that resemble West Nile virus. Furthermore, the WNV structural protein was expressed in Escherichia coli as a series of overlapping 20-mer peptides fused to a carrier-protein. Cytokine-based ELISPOT assays using these purified peptides revealed positive WNV-specific T cell responses to peptides within the different domains of the E-protein
The Prevalence of Coxiella burnetii in Hard Ticks in Europe and Their Role in Q Fever Transmission RevisitedâA Systematic Review
The zoonosis Q fever is caused by the obligate intracellular bacterium Coxiella burnetii.
Besides the main transmission route via inhalation of contaminated aerosols, ticks are
discussed as vectors since the first isolation of the pathogen from a Dermacentor
andersonii tick. The rare detection of C. burnetii in ticks and the difficult differentiation
of C. burnetii from Coxiella-like endosymbionts (CLEs) are questioning the relevance of
ticks in the epidemiology of Q fever. In this review, literature databases were systematically
searched for recent prevalence studies concerning C. burnetii in ticks in Europe and
experimental studies evaluating the vector competence of tick species. A total of 72
prevalence studies were included and evaluated regarding DNA detection methods and
collectionmethods, country, and tested tick species. Specimens ofmore than 25 different
tick species were collected in 23 European countries. Overall, an average prevalence of
4.8% was determined. However, in half of the studies, no Coxiella-DNA was detected. In
Southern European countries, a significantly higher prevalence was observed, possibly
related to the abundance of different tick species here, namely Hyalomma spp. and
Rhipicephalus spp. In comparison, a similar proportion of studies used ticks sampled by
flagging and dragging or tick collection from animals, under 30% of the total tick samples
derived from the latter. There was no significant difference in the various target genes
used for the molecular test. In most of the studies, no distinction was made between
C. burnetii and CLEs. The application of specific detection methods and the confirmation
of positive results are crucial to determine the role of ticks in Q fever transmission. Only
two studies were available, which assessed the vector competence of ticks for C. burnetii
in the last 20 years, demonstrating the need for further research
Specific detection of dengue and Zika virus antibodies using envelope proteins with mutations in the conserved fusion loop
Detection of antibodies is widely used for the diagnosis of infections with arthropod-borne flaviviruses including dengue (DENV) and Zika virus (ZIKV). Due to the emergence of ZIKV in areas endemic for DENV, massive co-circulation is observed and methods to specifically diagnose these infections and differentiate them from each other are mandatory. However, serological assays for flaviviruses in general, and for DENV and ZIKV in particular, are compromised by the high degree of similarities in their proteins which can lead to cross-reacting antibodies and false-positive test results. Cross-reacting flavivirus antibodies mainly target the highly conserved fusion loop (FL) domain in the viral envelope (E-) protein, and we and others have shown previously that recombinant E-proteins bearing FL-mutations strongly reduce cross-reactivity. Here we investigate whether such mutant E-proteins can be used to specifically detect antibodies against DENV and ZIKV in an ELISA-format. IgM antibodies against DENV and ZIKV virus were detected with 100% and 94.2% specificity and 90.7% and 87.5% sensitivity, respectively. For IgG the mutant E-proteins showed cross-reactivity, which was overcome by pre-incubation of the sera with the heterologous antigen. This resulted in specificities of 97.1% and 97.9% and in sensitivities of 100% and 100% for the DENV and ZIKV antigens, respectively. Our results suggest that E-proteins bearing mutations in the FL-domain have a high potential for the development of serological DENV and ZIKV tests with high specificity
Rapid decline of Zika virus NS1 antigen-specific antibody responses, northeastern Brazil
Zika virus (ZIKV) is a positive-stranded RNA virus within the Flaviviridae family. After decades of circulation in Asia, ZIKV was introduced to Brazil in 2014â2015, associated with a rise in congenital malformations. Unlike the genetically related dengue virus (DENV), ZIKV constitutes only one serotype. Although assumed that ZIKV infection may engender lifelong immunity, the long-term kinetics of ZIKV antibody responses are unclear. We assessed long-term kinetics of ZIKV NS1-IgG response in 144 individuals from 3 different subpopulations: HIV patients, tuberculosis patients and healthy individuals first tested in 2016 and retested 1.5â2 years after the 2015â2016 ZIKV epidemic in Salvador de Bahia, Brazil, using a widely distributed NS1-based commercial ELISA. The seropositivity in 2016 reached 59.0% (85/144, 95% confidence interval (CI) 50.7â66.7%), and decreased to 38.6% (56/144, CI 31.3â47.0%) 1.5â2 years later. In addition, the median ZIKV NS1-ELISA reactivity for individuals that remained positive in both timepoints significantly decreased from a ratio of 4.4 (95% CI 3.8â5.0) to 1.6 (95% CI 1.6â1.9) over the 2-year interval (Z: â 6.1; pâ<â0.001) irrespective of the subpopulation analyzed. Initial 2016 DENV antibody response was non-significant between groups, suggesting comparable DENV background. The high 20.6% seroreversion suggest that widely used serologic tests may fail to account a considerable proportion of past ZIKV infections in flavivirus endemic countries. In addition, ZIKV immunity might be shorter-lived than previously thought, which may contribute to local ZIKV resurgence once individual immune responses wane sufficiently to reduce community protective immunity in addition to birth and migration
Low-Energy Electron Irradiation Efficiently Inactivates the Gram-Negative Pathogen Rodentibacter pneumotropicusâA New Method for the Generation of Bacterial Vaccines with Increased Efficacy
Bacterial pathogens cause severe infections worldwide in livestock and in humans, and antibiotic resistance further increases the importance of prophylactic vaccines. Inactivated bacterial vaccines (bacterins) are usually produced via incubation of the pathogen with chemicals such as formaldehyde, which is time consuming and may cause loss of immunogenicity due to the modification of structural components. We evaluated low-energy electron irradiation (LEEI) as an alternative method to generate a bacterin. Rodentibacter pneumotropicus, an invasive Gram-negative murine pathogen, was inactivated with LEEI and formaldehyde. LEEI resulted in high antigen conservation, and LPS activity was significantly better maintained when compared with formaldehyde treatment. Immunization of mice with LEEI-inactivated R. pneumotropicus elicited a strong immune response with no detectable bacterial burden upon sublethal challenge. The results of this study suggest the inactivation of bacteria with LEEI as an alternative, fast and efficient method to generate bacterial vaccines with increased efficacy
Immunogenicity and protection efficacy of a naked self-replicating mRNA-based Zika virus vaccine
To combat emerging infectious diseases like Zika virus (ZIKV), synthetic messenger RNAs (mRNAs) encoding viral antigens are very attractive as they allow a rapid, generic, and flexible production of vaccines. In this work, we engineered a self-replicating mRNA (sr-mRNA) vaccine encoding the pre-membrane and envelope (prM-E) glycoproteins of ZIKV. Intradermal electroporation of as few as 1 ”g of this mRNA-based ZIKV vaccine induced potent humoral and cellular immune responses in BALB/c and especially IFNAR1-/- C57BL/6 mice, resulting in a complete protection of the latter mice against ZIKV infection. In wild-type C57BL/6 mice, the vaccine resulted in very low seroconversion rates and antibody titers. The potency of the vaccine was inversely related to the dose of mRNA used in wild-type BALB/c or C57BL/6 mice, as robust type I interferon (IFN) response was determined in a reporter mice model (IFN-ÎČ+/ÎÎČ-luc). We further investigated the inability of the sr-prM-E-mRNA ZIKV vaccine to raise antibodies in wild-type C57BL/6 mice and found indications that type I IFNs elicited by this naked sr-mRNA vaccine might directly impede the induction of a robust humoral response. Therefore, we assume that the efficacy of sr-mRNA vaccines after intradermal electroporation might be increased by strategies that temper their inherent innate immunogenicity
Low Energy Electron Irradiation Is a Potent Alternative to Gamma Irradiation for the Inactivation of (CAR-)NK-92 Cells in ATMP Manufacturing
Background: With increasing clinical use of NK-92 cells and their CAR-modified
derivatives in cancer immunotherapy, there is a growing demand for efficient
production processes of these âoff-the-shelfâ therapeutics. In order to ensure safety
and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be
inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently,
we showed proof of concept that low energy electron irradiation (LEEI) is a new method for
NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster
reaction time, a more reproducible dose rate and much less requirements on radiation
shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation
yielding cells with highly maintained cytotoxic effector function.
Methods: Effectiveness and efficiency of LEEI and gamma irradiation were analyzed using
NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively
characterized and compared to gamma-irradiated cells via flow cytometry, cytotoxicity
assays, and comet assays, amongst others.
Results: Our results show that both irradiation methods caused a progressive decrease
in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NKmediated
specific lysis of tumor cells was maintained at stable levels for three days postirradiation,
with a trend towards higher activities after LEEI treatment as compared to
gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage
and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition,
transcriptomic analysis of irradiated cells revealed approximately 12-fold more
differentially expressed genes two hours after gamma irradiation, compared to LEEI.
Analysis of surface molecules revealed an irradiation-induced decrease in surface
expression of CD56, but no changes in the levels of the activating receptors NKp46,
NKG2D, or NKp30.
Conclusions: The presented data show that LEEI inactivates (CAR-)NK-92 cells as
efficiently as gamma irradiation, but with less impact on the overall gene expression. Due
to logistic advantages, LEEI might provide a superior alternative for the manufacture of
(CAR-)NK-92 cells for clinical application
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