a new coronavirus emerged in China and caused an acute respiratory disease now known as coronavirus disease 2019 (COVID-19) (1). The virus was identified to be a betacoronavirus related to severe acute respiratory syndrome coronavirus (SARS-CoV) and thus was named SARS-CoV-2 (2). In <2 decades, this virus is the third known coronavirus to cross the species barrier and cause severe respiratory infections in humans after SARS-CoV in 2003 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, yet with unprecedented spread compared with the earlier 2 viruses.Because of the rapid increase in number of cases and uncontrolled and vast spread worldwide, the World Health Organization has declared SARS-CoV-2 a pandemic. As of March 14, 2020, the virus had infected >130,000 persons in 122 countries, 3.7% of whom had died. (3). Rapid identification of the etiology and sharing of the genetic sequence of the virus, followed by international collaborative efforts initiated because of emergence of SARS-CoV-2, has led to rapid availability of real-time PCR diagnostic assays that support case ascertainment and tracking of the outbreak (4). Availability of these assays has helped in patient detection and efforts to contain the virus. However, validated serologic assays are still lacking and are urgently needed.Validated serologic assays are crucial for patient contact tracing, identifying the viral reservoir hosts, and epidemiologic studies. Epidemiologic studies are urgently needed to help uncover the burden of disease, in particular the rate of asymptomatic infections, and to get better estimates on illness and death. In ad-

A new coronavirus, SARS-CoV-2, has recently emerged to cause a human pandemic.Whereas molecular diagnostic tests were rapidly developed, serologic assays are still lacking, yet urgently needed. Validated serologic assays are important for contact tracing, identifying the viral reservoir and epidemiological studies. Here, we developed serological assays for the detection of SARS-CoV-2 neutralizing, spike-and nucleocapsid-specific antibodies. Using serum samples from patients with PCR-confirmed infections of SARS-CoV-2, other coronaviruses, or other respiratory pathogenic infections, we validated and tested various antigens in different in-house and commercial ELISAs. We demonstrate that most PCRconfirmed SARS-CoV-2 infected individuals seroconverted, as revealed by sensitive and specific in-house ELISAs. We found that commercial S1 IgG or IgA ELISAs were of lower specificity while sensitivity varied between the two, with IgA showing higher sensitivity. Overall, the validated assays described here can be instrumental for the detection of SARS-CoV-2-specific antibodies for diagnostic, seroepidemiological and vaccine evaluation studies.

The emergence and rapid spread of SARS-CoV-2 variants may impact vaccine efficacy significantly. The Omicron variant termed BA.2, which differs substantially from BA.1 based on genetic sequence, is currently replacing BA.1 in several countries, but its antigenic characteristics have not yet been assessed. Here, we used antigenic cartography to quantify and visualize antigenic differences between early SARS-CoV-2 variants (614G, Alpha, Beta, Gamma, Zeta, Delta and Mu) using hamster antisera obtained after primary infection. We first verified that the choice of the cell line for the neutralization assay did not affect the topology of the map substantially. Antigenic maps generated using pseudotyped SARS-CoV-2 on the widely used VeroE6 cell line and the human airway cell line Calu-3 generated similar maps. Maps made using authentic SARS-CoV-2 on Calu-3 cells also closely resembled those generated with pseudotyped viruses. The antigenic maps revealed a central cluster of SARS-CoV-2 variants, which grouped based on mutual spike mutations. Whereas these early variants are antigenically similar, clustering relatively close to each other in antigenic space, Omicron BA.1 and BA.2 have evolved as two distinct antigenic outliers. Our data show that BA.1 and BA.2 both escape vaccine-induced antibody responses as a result of different antigenic characteristics. Thus, antigenic cartography could be used to assess antigenic properties of future SARS-CoV-2 variants of concern that emerge and to decide on the composition of novel spike-based (booster) vaccines.

Middle East respiratory syndrome coronavirus (MERS-CoV) infections in humans can cause asymptomatic to fatal lower respiratory lung disease. Despite posing a probable risk for virus transmission, asymptomatic to mild infections can go unnoticed; a lack of seroconversion among some PCR-confirmed cases has been reported. We found that a MERS-CoV spike S1 protein–based ELISA, routinely used in surveillance studies, showed low sensitivity in detecting infections among PCR-confirmed patients with mild clinical symptoms and cross-reactivity of human coronavirus OC43–positive serum samples. Using in-house S1 ELISA and protein microarray, we demonstrate that most PCR-confirmed MERS-CoV case-patients with mild infections seroconverted; nonetheless, some of these samples did not have detectable levels of virus-neutralizing antibodies. The use of a sensitive and specific serologic S1-based assay can be instrumental in the accurate estimation of MERS-CoV prevalence.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the cause of coronavirus disease 2019 . Little is known about the interplay between pre-existing immunity towards endemic seasonal coronaviruses and the development of a SARS-CoV-2-specific IgG response. We investigated the kinetics, breadth, magnitude and level of cross-reactivity of IgG antibodies against SARS-CoV-2 and heterologous seasonal and epidemic coronaviruses at the clonal level in mild and severe COVID-19 patients and disease control patients. Antibody reactivity towards nucleocapsid and spike antigens was assessed and correlated to SARS-CoV-2 neutralization. COVID-19 patients mounted a mostly type-specific SARS-CoV-2 response. Additionally, IgG clones directed against seasonal coronavirus were boosted in patients with severe COVID-19. These boosted clones showed limited cross-reactivity and did not neutralize SARS-CoV-2. These findings support a boost of poorly protective coronavirusspecific antibodies in COVID-19 patients that correlates with disease severity, revealing original antigenic sin.