Liposome-based high-throughput and point-of-care assays toward the quick, simple, and sensitive detection of neutralizing antibodies against SARS-CoV-2 in patient sera.

The emergence of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) in 2019 caused an increased interest in neutralizing antibody tests to determine the immune status of the population. Standard live-virus-based neutralization assays such as plaque-reduction assays or pseudovirus neutralization tests cannot be adapted to the point-of-care (POC). Accordingly, tests quantifying competitive binding inhibition of the angiotensin-converting enzyme 2 (ACE2) receptor to the receptor-binding domain (RBD) of SARS-CoV-2 by neutralizing antibodies have been developed. Here, we present a new platform using sulforhodamine B encapsulating liposomes decorated with RBD as foundation for the development of both a fluorescent, highly feasible high-throughput (HTS) and a POC-ready neutralizing antibody assay. RBD-conjugated liposomes are incubated with serum and subsequently immobilized in an ACE2-coated plate or mixed with biotinylated ACE2 and used in test strip with streptavidin test line, respectively. Polyclonal neutralizing human antibodies were shown to cause complete binding inhibition, while S309 and CR3022 human monoclonal antibodies only caused partial inhibition, proving the functionality of the assay. Both formats, the HTS and POC assay, were then tested using 20 sera containing varying titers of neutralizing antibodies, and a control panel of sera including prepandemic sera and reconvalescent sera from respiratory infections other than SARS-CoV-2. Both assays correlated well with a standard pseudovirus neutralization test (r = 0.847 for HTS and r = 0.614 for POC format). Furthermore, excellent correlation (r = 0.868) between HTS and POC formats was observed. The flexibility afforded by liposomes as signaling agents using different dyes and sizes can hence be utilized in the future for a broad range of multianalyte neutralizing antibody diagnostics.

A systematic approach for the identification of novel, serologically reactive recombinant Varicella-Zoster Virus (VZV) antigens.

BACKGROUND: Varicella-Zoster virus causes chickenpox upon primary infection and shingles after reactivation. Currently available serological tests to detect VZV-specific antibodies are exclusively based on antigens derived from VZV-infected cells. RESULTS: We present a systematic approach for the identification of novel, serologically reactive VZV antigens. Therefore, all VZV open reading frames were cloned into a bacterial expression vector and checked for small scale recombinant protein expression. Serum profiling experiments using purified VZV proteins and clinically defined sera in a microarray revealed 5 putative antigens (ORFs 1, 4, 14, 49, and 68). These were rearranged in line format and validated with pre-characterized sera. CONCLUSIONS: The line assay confirmed the seroreactivity of the identified antigens and revealed its suitability for VZV serodiagnostics comparable to commercially available VZV-ELISA. Recombinant ORF68 (gE) proved to be an antigen for high-confidence determination of VZV serostatus. Furthermore, our data suggest that a serological differentiation between chickenpox and herpes zoster may be possible by analysis of the IgM-portfolio against individual viral antigens.

Serologic analysis of the IgG antibody response in children with varicella zoster virus wild-type infection and vaccination.

INTRODUCTION: In contrast to varicella zoster virus (VZV) primary infection, VZV vaccination does not seem to provide lifelong immunity against varicella. Because more people get vaccinated every year, the development of sensitive serological test systems for the detection of protective anti-VZV IgG will become important in the future. METHODS: We have previously developed a novel VZV line assay based on 5 different recombinant VZV antigens. In this study, we compared this novel assay with a commercially available glycoprotein enzyme immunoassay (RIDASCREEN VZV IgG) in detecting anti-VZV IgG of children with previous varicella infection and VZV vaccination. RESULTS: One hundred twenty-five children were included in this study, 72 with a history of varicella infection and 53 with VZV vaccination. Both assays detected anti-VZV IgG antibodies in both study groups with similar sensitivities. The VZV line assay revealed striking differences in the anti-VZV IgG composition against the VZV open reading frames, 4, 14 and 49, between both study groups, indicating that wild-type varicella infection causes a more diverse immune response against VZV than does vaccination. The exploitation of these results enabled the discrimination of both study groups with a sensitivity of 0.93 and a specificity of 0.83, indicating that the serologic differentiation of children with previous varicella infection and VZV vaccination might be possible. CONCLUSION: The VZV line assay enables the detection of anti-VZV IgG with sensitivities comparable to glycoprotein enzyme immunoassays and might be suitable for the serologic discrimination between children with a history of varicella infection and VZV vaccination.