Recent Developments in SARS-CoV-2 Neutralizing Antibody Detection Methods.

The ongoing Coronavirus disease 19 pandemic has likely changed the world in ways not seen in the past. Neutralizing antibody (NAb) assays play an important role in the management of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) outbreak. Using these tools, we can assess the presence and duration of antibody-mediated protection in naturally infected individuals, screen convalescent plasma preparations for donation, test the efficacy of immunotherapy, and analyze NAb titers and persistence after vaccination to predict vaccine-induced protective effects. This review briefly summarizes the various methods used for the detection of SARS-CoV-2 NAbs and compares their advantages and disadvantages to facilitate their development and clinical application.

Development and evaluation of a new immunohistochemistry-based test for the detection of rabies virus neutralizing antibodies.

Rabies claims about 55,000 human lives and many hundreds of thousands of livestock every year, worldwide. Despite a heavy disease burden, laboratory facilities to diagnose the infection remain scarce in most countries of the developing world where the disease is endemic. Rapid Fluorescent Focus Inhibition Test (RFFIT) and Fluorescent Antibody Virus Neutralization Test (FAVN) are the common tests done in the rabies diagnostic laboratories to detect and quantitate Rabies Virus Neutralizing Antibodies (RVNA). RFFIT is most often employed in confirming seroconversion following prophylactic vaccination, and to aid ante-mortem diagnosis in suspected cases of rabies. Though this remains one of the most sought-after diagnostic services in rabies laboratories, the requirements for expensive anti-rabies fluorochrome antibody conjugate and a fluorescent microscope restrict its performance to only a few reference laboratories. Cost-effective laboratory diagnostic methods employing affordable technology are a need of the hour in the rabies-endemic countries. In this study we have developed a new immunohistochemistry-based neutralization test and extensively evaluated it along with RFFIT. One hundred and 20 human serum samples collected after post-exposure vaccination were subjected to both the tests for determining RVNA titers. The results obtained with the new test correlated significantly with those of RFFIT. Further validation of the inter- and intra- assay precision, lower limit of quantification (LLOQ) and specificity was also performed. The best correlation between the 2 methods, however, was observed only when the RVNA concentrations in the samples were>20 IU/mL. Overall, the immunohistrochemistry-based neutralization test yielded satisfactory results. We suggest that it might serve as a cost-effective alternative to RFFIT in low-resource settings in the developing countries.

LabKey Server NAb: a tool for analyzing, visualizing and sharing results from neutralizing antibody assays.

BACKGROUND: Multiple types of assays allow sensitive detection of virus-specific neutralizing antibodies. For example, the extent of antibody neutralization of HIV-1, SIV and SHIV can be measured in the TZM-bl cell line through the degree of luciferase reporter gene expression after infection. In the past, neutralization curves and titers for this standard assay have been calculated using an Excel macro. Updating all instances of such a macro with new techniques can be unwieldy and introduce non-uniformity across multi-lab teams. Using Excel also poses challenges in centrally storing, sharing and associating raw data files and results. RESULTS: We present LabKey Server's NAb tool for organizing, analyzing and securely sharing data, files and results for neutralizing antibody (NAb) assays, including the luciferase-based TZM-bl NAb assay. The customizable tool supports high-throughput experiments and includes a graphical plate template designer, allowing researchers to quickly adapt calculations to new plate layouts. The tool calculates the percent neutralization for each serum dilution based on luminescence measurements, fits a range of neutralization curves to titration results and uses these curves to estimate the neutralizing antibody titers for benchmark dilutions. Results, curve visualizations and raw data files are stored in a database and shared through a secure, web-based interface. NAb results can be integrated with other data sources based on sample identifiers. It is simple to make results public after publication by updating folder security settings. CONCLUSIONS: Standardized tools for analyzing, archiving and sharing assay results can improve the reproducibility, comparability and reliability of results obtained across many labs. LabKey Server and its NAb tool are freely available as open source software at http://www.labkey.com under the Apache 2.0 license. Many members of the HIV research community can also access the LabKey Server NAb tool without installing the software by using the Atlas Science Portal (https://atlas.scharp.org). Atlas is an installation of LabKey Server.

Structural basis of influenza virus neutralization.

Although seasonal influenza vaccines play a valuable role in reducing the spread of virus at the population level, ongoing viral evolution to evade immune responses remains problematic. No current vaccines elicit enduring protection in the face of emerging and re-emerging influenza viruses that are rapidly undergoing antigenic drift. Eliciting broadly cross-neutralizing antibody (nAb) responses against influenza virus is a crucial goal for seasonal and pandemic influenza vaccine preparation. Recent three-dimensional structure information obtained from crystallization of influenza antigens in complex with nAbs has provided a framework for interpreting antibody-based viral neutralization that should aid in the design of vaccine immunogens. Here, we will review current knowledge of the structure-based mechanisms contributing to the neutralization and neutralization escape of influenza viruses. We will also explore the potential for this structure-based approach to overcome the obstacles in developing the highly desired "universal" influenza vaccine.

Basic rationale, current methods and future directions for molecular typing of human enterovirus.

Enterovirus is a genus of the Picornaviridae family including more than 80 serotypes belonging to four species designed Human enterovirus A to D. The antigens of the structural proteins support the subdivision of enteroviruses into multiple serotypes. Comparative phylogeny based on molecular typing methods has been of great help to classify former and new types of enterovirus, and to investigate the diversity of enteroviruses and the evolutionary mechanisms involved in their diversity. By now, molecular typing methods of enterovirus rely mainly on the sequencing of an amplicon targeting a variable part of the region coding for the capsid proteins (VP1 and, alternatively, VP2 or VP4), either from a strain recovered by cell culture or, more recently, by direct amplification of a clinical or environmental specimen. In the future, microarrays are thought to play a major role in enterovirus typing and in the analysis of the determinants of virulence that support the puzzling diversity of the pathological conditions associated with human infection by these viruses.

ELISA as a possible alternative to the neutralization test for evaluating the immune response to poliovirus vaccines.

This review describes several enzyme-linked immunosorbent assay (ELISA) techniques proposed to replace the neutralization test for detecting neutralization-relevant antibodies to polioviruses in recipients of inactivated poliovirus vaccine and oral poliovirus vaccine, and for seroepidemiologic studies. Comparisons of results from ELISA and the neutralization test suggest that ELISA variants, based on the principle of blocking or binding inhibition that emulate the neutralization test, might offer an alternative to the neutralization test. However, to replace the neutralization test with ELISA would first require extensive studies with very large numbers of serum samples, including sera having low titers of neutralizing antibodies, in order to obtain reliable and statistically sound validation.