Unexpected detection of SARS-CoV-2 antibodies in the prepandemic period in Italy.

There are no robust data on the real onset of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and spread in the prepandemic period worldwide. We investigated the presence of SARS-CoV-2 receptor-binding domain (RBD)-specific antibodies in blood samples of 959 asymptomatic individuals enrolled in a prospective lung cancer screening trial between September 2019 and March 2020 to track the date of onset, frequency, and temporal and geographic variations across the Italian regions. SARS-CoV-2 RBD-specific antibodies were detected in 111 of 959 (11.6%) individuals, starting from September 2019 (14%), with a cluster of positive cases (>30%) in the second week of February 2020 and the highest number (53.2%) in Lombardy. This study shows an unexpected very early circulation of SARS-CoV-2 among asymptomatic individuals in Italy several months before the first patient was identified, and clarifies the onset and spread of the coronavirus disease 2019 (COVID-19) pandemic. Finding SARS-CoV-2 antibodies in asymptomatic people before the COVID-19 outbreak in Italy may reshape the history of pandemic.

Comparative analyses of SARS-CoV-2 binding (IgG, IgM, IgA) and neutralizing antibodies from human serum samples.

A newly identified coronavirus, named SARS-CoV-2, emerged in December 2019 in Hubei Province, China, and quickly spread throughout the world; so far, it has caused more than 49.7 million cases of disease and 1,2 million deaths. The diagnosis of SARS-CoV-2 infection is currently based on the detection of viral RNA in nasopharyngeal swabs by means of molecular-based assays, such as real-time RT-PCR. Furthermore, serological assays detecting different classes of antibodies constitute an excellent surveillance strategy for gathering information on the humoral immune response to infection and the spread of the virus through the population. In addition, it can contribute to evaluate the immunogenicity of novel future vaccines and medicines for the treatment and prevention of COVID-19 disease. The aim of this study was to determine SARS-CoV-2-specific antibodies in human serum samples by means of different commercial and in-house ELISA kits, in order to evaluate and compare their results first with one another and then with those yielded by functional assays using wild-type virus. It is important to identify the level of SARS-CoV-2-specific IgM, IgG and IgA antibodies in order to predict human population immunity, possible cross-reactivity with other coronaviruses and to identify potentially infectious subjects. In addition, in a small sub-group of samples, a subtyping IgG ELISA has been performed. Our findings showed a notable statistical correlation between the neutralization titers and the IgG, IgM and IgA ELISA responses against the receptor-binding domain of the spike protein. Thus confirming that antibodies against this portion of the virus spike protein are highly neutralizing and that the ELISA Receptor-Binding Domain-based assay can be used as a valid surrogate for the neutralization assay in laboratories that do not have biosecurity level-3 facilities.

Evaluation of SARS-CoV-2 neutralizing antibodies using a CPE-based colorimetric live virus micro-neutralization assay in human serum samples.

The micro-neutralization assay is a fundamental test in virology, immunology, vaccine assessment, and epidemiology studies. Since the SARS-CoV-2 outbreak at the end of December 2019 in China, it has become extremely important to have well-established and validated diagnostic and serological assays for this new emerging virus. Here, we present a micro-neutralization assay with the use of SARS-CoV-2 wild type virus with two different methods of read-out. We evaluated the performance of this assay using human serum samples taken from an Italian seroepidemiological study being performed at the University of Siena, along with the human monoclonal antibody CR3022 and some iper-immune animal serum samples against Influenza and Adenovirus strains. The same panel of human samples have been previously tested in enzyme-linked immunosorbent assay (ELISA) as a pre-screening. Positive, borderline, and negative ELISA samples were evaluated in neutralization assay using two different methods of read-out: subjective (by means of an inverted optical microscope) and objective (by means of a spectrophotometer). Our findings suggest that at least 50% of positive ELISA samples are positive in neutralization as well, and that method is able to quantify different antibody concentrations in a specific manner. Taken together, our results confirm that the colorimetric cytopathic effect-based microneutralization assay could be used as a valid clinical test method for epidemiological and vaccine studies.

Comparison of influenza-specific neutralizing antibody titers determined using different assay readouts and hemagglutination inhibition titers: good correlation but poor agreement.

Determination of influenza-specific antibody titers is commonly done using the hemagglutination inhibition assay (HAI) and the viral microneutralization assay (MN). Both assays are characterized by high intra- and inter-laboratory variability. The HAI assay offers little opportunity for standardization. For the MN assay, variability might be due to the use of different assay protocols employing different readouts. We therefore aimed at investigating which of the MN assay readout methods currently in use would be the most suitable choice for a standardized MN assay that could serve as a substitute for the HAI assay. For this purpose, human serum samples were tested for the presence of influenza specific neutralizing antibodies against A/California/7/09 H1N1 (49 sera) or A/Hong Kong/4801/2014 (50 sera) using four different infection readout methods for the MN assay (cytopathic effect, hemagglutination, ELISA, RT qPCR) and using the HAI assay. The results were compared by correlation analysis and by determining the level of agreement before and after normalization to a standard serum. Titers as measured by the 4 MN assay readouts showed good correlation, with high Person's r for most comparisons. However, agreement between nominal titers varied with readouts compared and virus strain used. In addition, Pearson's correlation of MN titers with HAI titers was high but agreement of nominal titers was moderate and the average difference between the readings of two assays (bias) was virus strain-dependent. Normalization to a standard serum did not result in better agreement of assay results. Our study demonstrates that different MN readouts result in nominally different antibody titers. Accordingly, the use of a common and standardized MN assay protocol will be crucial to minimize inter-laboratory variability. Based on reproducibility, cost effectiveness and unbiased assessment of results we elected the MN assay with ELISA readout as most suitable for a possible replacement of the HAI assay.