Constitutive IFNα Protein Production in Bats

Bats are the only mammals with self-powered flight and account for 20% of all extant mammalian diversity. In addition, they harbor many emerging and reemerging viruses, including multiple coronaviruses, several of which are highly pathogenic in other mammals, but cause no disease in bats. How this symbiotic relationship between bats and viruses exists is not yet fully understood. Existing evidence supports a specific role for the innate immune system, in particular type I interferon (IFN) responses, a major component of antiviral immunity. Previous studies in bats have shown that components of the IFN pathway are constitutively activated at the transcriptional level. In this study, we tested the hypothesis that the type I IFN response in bats is also constitutively activated at the protein level. For this, we utilized highly sensitive Single Molecule (Simoa) digital ELISA assays, previously developed for humans that we adapted to bat samples. We prospectively sampled four non-native chiroptera species from French zoos. We identified a constitutive expression of IFNα protein in the circulation of healthy bats, and concentrations that are physiologically active in humans. Expression levels differed according to the species examined, but were not associated with age, sex, or health status suggesting constitutive IFNα protein expression independent of disease. These results confirm a unique IFN response in bat species that may explain their ability to coexist with multiple viruses in the absence of pathology. These results may help to manage potential zoonotic viral reservoirs and potentially identify new anti-viral strategies.

Constitutive IFNα protein production in bats

Bats are the only mammals with self-powered flight and account for 20% of all extant mammalian diversity. In addition, they harbor many emerging and reemerging viruses, including multiple coronaviruses, several of which are highly pathogenic in other mammals, but cause no disease in bats. How this relationship between bats and viruses exists is not yet fully understood. Existing evidence supports a specific role for the innate immune system, in particular type I interferon (IFN) responses, a major component of antiviral immunity. Previous studies in bats have shown that components of the IFN pathway are constitutively activated at the transcriptional level. In this study, we tested the hypothesis that the type I IFN response in bats is also constitutively activated at the protein level. For this we utilized highly sensitive Single Molecule (Simoa) digital ELISA assays, previously developed for humans that we adapted to bat samples. We prospectively sampled four non-native chiroptera species from French zoos. We identified a constitutive expression of IFNα protein in the circulation of healthy bats, and concentrations that are physiologically active in humans. Expression levels differed according to the species examined, but was not associated with age, sex, or health status suggesting constitutive IFNα protein expression independent of disease. These results confirm a unique IFN response in bat species that may explain their ability to coexist with multiple viruses in the absence of pathology. These results may help to manage potential zoonotic viral reservoirs and potentially identify new anti-viral strategies.

Differential levels of IFNα subtypes in autoimmunity and viral infection

Type I interferons are essential for host response to viral infections, while dysregulation of their response can result in autoinflammation or autoimmunity. Among IFNα (alpha) responses, 13 subtypes exist that signal through the same receptor, but have been reported to have different effector functions. However, the lack of available tools for discriminating these closely related subtypes, in particular at the protein level, has restricted the study of their differential roles in disease. We developed a digital ELISA with specificity and high sensitivity for the IFNα2 subtype. Application of this assay, in parallel with our previously described pan-IFNα assay, allowed us to study different IFNα protein responses following cellular stimulation and in diverse patient cohorts. We observed different ratios of IFNα protein responses between viral infection and autoimmune patients. This analysis also revealed a small percentage of autoimmune patients with high IFNα2 protein measurements but low pan-IFNα measurements. Correlation with an ISG score and functional activity showed that in this small sub group of patients, IFNα2 protein measurements did not reflect its biological activity. This unusual phenotype was partly explained by the presence of anti-IFNα auto-antibodies in a subset of autoimmune patients. This study reports ultrasensitive assays for the study of IFNα proteins in patient samples and highlights the insights that can be obtained from the use of multiple phenotypic readouts in translational and clinical studies.

Ultrasensitive detection of p24 in plasma samples from people with primary and chronic HIV-1 infection

HIV-1 Gag p24 has long been identified as an informative biomarker of HIV replication, disease progression and therapeutic efficacy, but the lower sensitivity of immunoassays in comparison to molecular tests and the interference with antibodies in chronic HIV infection limits its application for clinical monitoring. The development of ultrasensitive protein detection technologies may help overcoming these limitations. Here we evaluated whether immune-complex dissociation combined with ultrasensitive digital ELISA Simoa technology could be used to quantify p24 in plasma samples from people with HIV-1 infection. We found that, among different immune-complex dissociation methods, only acid-mediated dissociation was compatible with ultrasensitive p24 quantification by digital ELISA, strongly enhancing p24 detection at different stages of HIV-1 infection. We show that ultrasensitive p24 levels correlated positively with plasma HIV-RNA and HIV-DNA and negatively with CD4+ T cells in the samples from people with primary and chronic HIV-1 infection. In addition, p24 levels also correlated with plasma D-dimers and IFNα levels. P24 levels sharply decreased to undetectable levels after initiation of combined antiretroviral treatment (cART). However, we identified a group of people who, 48 weeks after cART initiation, had detectable p24 levels despite having undetectable viral loads. These people had different virologic and immunologic baseline characteristics when compared with people who had undetectable p24 after cART. These results demonstrate that ultrasensitive p24 analysis provides an efficient and robust mean to monitor p24 antigen in plasma samples from people with HIV-1 infection, including during antiretroviral treatment, and may provide complementary information to other commonly used biomarkers.

A method for detecting IL-6 in serum of patients with uremia

As the gold standard of protein detection, enzyme-linked immunosorbent assay (ELISA) is widely used in medical treatment and biology. Here, we report a digital ELISA method that combines fluorescence-coded magnetic beads with micropore arrays to effectively improve the accuracy of the detection. Fluorescence coded magnetic beads were used as solid support of ELISA, which were modified to specifically capture IL-6 in serum, and then combined with galactosidase to form a sandwich structure. These beads are then mixed with a fluorescent substrate and passed into a microfluidic chip. Under the action of gravity, the beads are trapped and isolated by an array of micropores in the chip. Combined with image recognition technology, the fluorescence intensity of micropores containing enzymes will increase rapidly. By mining image information, the IL-6 content in uremia patients can be detected with high precision.

Digital enzyme-linked immunosorbent assays with sub-attomolar detection limits based on low numbers of capture beads combined with high efficiency bead analysis

We report approaches to improve the sensitivity of digital ELISA up to 400-fold, enabling detection of proteins at subattomolar concentrations.

Transmembrane signaling on a protocell: Creation of receptor-enzyme chimeras for immunodetection of specific antibodies and antigens

It is known that digital counting of fluorescent signals generated in many small compartments can significantly improve the detection sensitivity of the enzyme-linked immunosorbent assay (ELISA). However, the reported digital ELISA systems need extensive washing steps to remove background signal, which hampers their performance. To tackle this problem, we developed a vesicle (Protocell) array wherein binding of an external protein analyte is coupled to signal amplification and intra-vesicular fluorescence readout. We chose β-glucuronidase (GUS) as a reporter enzyme as its function requires assembly of four subunits through dimerization of a pair of dimers that can be inhibited by a set of interface mutations. Using a thermostabilized GUS mutant IV-5, we screened out an interface mutant (M516K, F517W) to create IV5m - a mutant with high thermostability and activity conditional on induced dimerization. After tethering a short N-terminal tag and transmembrane (TM) sequences, the fusion protein was expressed by cell-free protein synthesis inside protocells. When a corresponding tag-specific antibody was applied outside of the protocells, a clear increase in GUS activity was observed inside vesicles by adding fluorescent substrate, probably due to spontaneous integration of the tagged TM protein into the vesicles and dimerization by the antibody bound to the displayed tag. Furthermore, using flow cytometry, quantitative digital read out was obtained by counting fluorescent protocells exposed to varying concentrations of external antibodies that included Trastuzumab. Additionally, through use of an anti-caffeine VHH-SpyCatcher fusion protein, caffeine could be detected using SpyTag-fused TM-IV5m protein expressed in protocells, suggesting utility of this platform for detection of diverse antigen types.