Validation of HAV biomarker 2A for differential diagnostic of hepatitis A infected and vaccinated individuals using multiplex serology

BackgroundWorldwide about 1.5 million clinical cases of hepatitis A virus (HAV) infections occur every year and increasingly countries are introducing HAV vaccination into the childhood immunization schedule with a single dose instead of the originally licenced two dose regimen. Diagnosis of acute HAV infection is determined serologically by anti-HAV-IgM detection using ELISA. Additionally anti-HAV-IgG can become positive during the early phase of symptoms, but remains detectable after infection and also after vaccination against HAV. Currently no serological marker allows the differentiation of HAV vaccinated individuals and those with a past infection with HAV. Such differentiation would greatly improve evaluation of vaccination campaigns and risk assessment of HAV outbreaks. Here we tested the HAV non-structural protein 2A, important for the capsid assembly, as a biomarker for the differentiation of the immune status in previously infected and vaccinated individuals.MethodsHAV antigens were recombinantly expressed as glutathione-S-transferase (GST) fusion proteins. Using glutathione tagged, magnetic fluorescent beads (Luminex®), the proteins were affinity purified and used in a multiplex serological assay. The multiplex HAV assay was validated using 381 reference sera in which the immune status HAV negative, vaccinated or infected was established using the Abbott ARCHITECT® HAVAb-IgM or IgG, the commercial HAV ELISA from Abnova and documentation in vaccination cards.ResultsHAV multiplex serology showed a sensitivity of 99% and specificity of 95% to detect anti-HAV IgG/IgM positive individuals. HAV biomarker 2A allowed the differentiation between previously infected and vaccinated individuals. HAV vaccinated individuals and previously infected individuals could be identified with 92% accuracy.ConclusionHAV biomarker 2A can be used to differentiate between previously HAV-vaccinated and naturally infected individuals. Within a multiplex serological approach this assay can provide valuable novel information in the context of outbreak investigations, longitudinal population based studies and evaluations of immunization campaigns.     

The senescence accelerated mouse prone 8 (SAMP8): A novel murine model for cardiac aging

Because cardiovascular disease remains the major cause of mortality and morbidity world-wide, there remains a compelling need for new insights and novel therapeutic avenues. In this regard, the senescence-accelerated mouse prone 8 (SAMP8) line is a particularly good model for studying the effects of aging on cardiovascular health. Accumulating evidence suggests that this model may shed light on age-associated cardiac and vascular dysfunction and disease. These animals manifest evidence of inflammation, oxidative stress and adverse cardiac remodeling that may recapitulate processes involved in human disease. Early alterations in oxidative damage promote endoplasmic reticulum stress to trigger apoptosis and cytokine production in this genetically susceptible mouse strain. Conversely, pharmacological treatments that reduce inflammation and oxidative stress improve cardiac function in these animals. Therefore, the SAMP8 mouse model provides an exciting opportunity to expand our knowledge of aging in cardiovascular disease and the potential identification of novel targets of treatment. Herein, we review the previous studies performed in SAMP8 mice that provide insight into age-related cardiovascular alterations.     

Humanized C3 Mouse: A Novel Accelerated Model of C3 Glomerulopathy

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A flexible split prime editor using truncated reverse transcriptase improves dual-AAV delivery in mouse liver

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An inducible transgenic mouse breast cancer model for the analysis of tumor antigen specific CD8+ T-cell responses

In Simian virus 40 (SV40) transgenic BALB/c WAP-T mice tumor development and progression is driven by SV40 tumor antigens encoded by inducible transgenes. WAP-T mice constitute a well characterized mouse model for breast cancer with strong similarities to the corresponding human disease. BALB/c mice mount only a weak cellular immune response against SV40 T-antigen (T-Ag). For studying tumor antigen specific CD8⁺ T-cell responses against transgene expressing cells, we created WAP-T_NP mice, in which the transgene additionally codes for the NP_118–126-epitope contained within the nucleoprotein of lymphocytic choriomeningitis virus (LCMV), the immune-dominant T-cell epitope in BALB/c mice. We then investigated in WAP-T_NP mice the immune responses against SV40 tumor antigens and the NP-epitope within the chimeric T-Ag/NP protein (T-Ag_NP). Analysis of the immune-reactivity against T-Ag in WAP-T and of T-Ag_NP in WAP-T_NP mice revealed that, in contrast to wild type (wt) BALB/c mice, WAP-T and WAP-T_NP mice were non-reactive against T-Ag. However, like wtBALB/c mice, WAP-T as well as WAP-T_NP mice were highly reactive against the immune-dominant LCMV NP-epitope, thereby allowing the analysis of NP-epitope specific cellular immune responses in WAP-T_NP mice. LCMV infection of WAP-T_NP mice induced a strong, LCMV NP-epitope specific CD8⁺ T-cell response, which was able to specifically eliminate T-Ag_NP expressing mammary epithelial cells both prior to tumor formation (i.e. in cells of lactating mammary glands), as well as in invasive tumors. Elimination of tumor cells, however, was only transient, even after repeated LCMV infections. Further studies showed that already non-infected WAP-T_NP tumor mice contained LCMV NP-epitope specific CD8⁺ T-cells, albeit with strongly reduced, though measurable activity. Functional impairment of these ‘endogenous’ NP-epitope specific T-cells seems to be caused by expression of the programmed death-1 protein (PD1), as anti-PD1 treatment of splenocytes from WAP-T_NP tumor mice restored their activity. These characteristics are similar to those found in many tumor patients and render WAP-T_NP mice a suitable model for analyzing parameters to overcome the blockade of immune checkpoints in tumor patients.     

Engineering of a recombinant trivalent single-chain variable fragment antibody directed against rabies virus glycoprotein G with improved neutralizing potency

Human and equine rabies immunoglobulins are currently available for passive immunization against rabies. However, these are hampered by the limited supply and some drawbacks. Advances in antibody engineering have led to overcome issues of clinical applications and to improve the protective efficacy. In the present study, we report the generation of a trivalent single-chain Fv (scFv50AD1-Fd), that recognizes the rabies virus glycoprotein, genetically fused to the trimerization domain of the bacteriophage T4 fibritin, termed ‘foldon’ (Fd). scFv50AD1-Fd was expressed as soluble recombinant protein in bacterial periplasmic space and purified through affinity chromatography. The molecular integrity and stability were analyzed by polyacrylamide gradient-gel electrophoresis, size-exclusion chromatography and incubation in human sera. The antigen-binding properties of the trimeric scFv were analyzed by direct and competitive-ELISA. Its apparent affinity constant was estimated at 1.4 ± 0.25 × 10⁹ M⁻¹ and was 75-fold higher than its monovalent scFv (1.9 ± 0.68 × 10⁷ M⁻¹). The scFv50AD1-Fd neutralized rabies virus in a standard in vitro and in vivo neutralization assay. We showed a high neutralization activity up to 75-fold compared with monovalent format and the WHO standard serum. The gain in avidity resulting from multivalency along with an improved biological activity makes the trivalent scFv50AD1-Fd construct an important reagent for rabies protection. The antibody engineering approach presented here may serve as a strategy for designing a new generation of anti-rabies for passive immunotherapy.     

Extracellular superoxide dismutase ameliorates house dust mite‐induced atopic dermatitis‐like skin inflammation and inhibits mast cell activation in mice

Extracellular superoxide dismutase (EC‐SOD) is an enzyme that catalyses the dismutation of superoxide anions. It has multiple functions, such as reactive oxygen species scavenging, anti‐angiogenic, anti‐inflammatory, antichemotatic and antitumor activities. Recently, we demonstrated that EC‐SOD inhibits ovalbumin‐induced allergic airway inflammation in mice. However, the anti‐allergic effect of EC‐SOD on skin tissue and the role of EC‐SOD in mast cells, which are important for allergic responses, have not been well studied. In this study, we investigated whether EC‐SOD can alleviate atopic dermatitis in mice and inhibit mast cell activation. Treatment with human recombinant EC‐SOD ameliorated house dust mite‐induced atopic dermatitis in mice. Furthermore, the levels of pro‐allergic cytokine gene expression and histamine release increased in EC‐SOD KO mast cells and decreased in EC‐SOD overexpressing mast cells, suggesting that EC‐SOD inhibits mast cell activation. Consistently, a passive cutaneous anaphylaxis experiment showed more blood leakage from EC‐SOD KO mouse ear skin, implying that the lack of EC‐SOD increases allergic responses. These results suggest that EC‐SOD inhibits mast cell activation and atopic dermatitis and that the loss of EC‐SOD causes more severe allergic responses, implying that EC‐SOD might be a good drug candidate for treatment of allergic disorders, such as atopic dermatitis.     

Mutations in the transcription factor FOXO1 mimic positive selection signals to promote germinal center B cell expansion and lymphomagenesis

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