key: cord-0989850-di2ajqj7 authors: Bauer, Georg title: The variability of the serological response to SARS corona virus‐2: Potential resolution of ambiguity through determination of avidity (functional affinity) date: 2020-07-07 journal: J Med Virol DOI: 10.1002/jmv.26262 sha: a24819b99becb56384fa6a38c16fc24c89b93efb doc_id: 989850 cord_uid: di2ajqj7 Data on the serological response towards SARS CoV‐2 in 16 recent reports were analyzed and a high degree of variability was shown. IgM responses were either found earlier than IgG, or together with IgG, later than IgG or were missing. Therefore, clear distinctions between early, intermediate and past infections are obviously not possible merely on the basis of IgM and IgG determinations. The review of publications on the serology of other virus groups shows that variable IgM responses can be found as well and therefore are not specific for SARS CoV‐2 infections. A model to explain this variability is proposed. The inclusion of avidity determination into regular diagnostic procedures has allowed to resolve such “atypical” serological constellations. The potential use of avidity determination for the diagnosis of Covid‐19, for risk assessment, epidemiological studies, analysis of cross reactions, as well as for the control of vaccination programs is suggested and discussed. This article is protected by copyright. All rights reserved. SARS corona virus-2 (SARS-CoV-2) is presently causing a pandemics with many cases of severe disease and death. This has a massive impact on daily life, the health system, economy, politics, science, education and international travel. World-wide, governments and NGOs try to develop strategies to counteract the pandemics and its consequences. The management of Covid-19 requires tools to i) diagnose or exclude SARS-CoV-2 infections in patients with respiratory symptoms; ii) define clinically asymptomatic as well as symptomatic persons that are infected with SARS-CoV-2, in order to prevent further spreading of the virus; iii) define persons that are seronegative for SARS-CoV-2 and therefore at risk for future SARS-CoV-2 infection; This article is protected by copyright. All rights reserved. iv) to define people with clinically asymptomatic SARS-CoV-2 infection and a positive immune response. It has to be clarified, whether these people are protected towards reinfection by SARS-CoV-2 and how long this possible protection lasts. There is an evolving consent that the detection of viral genomes through PCR, as well as the determination of specific antibody responses, will be required to answer the questions summarized above. Due to the characteristics of the viral infection and the resultant serological response, obviously none of these two approaches alone is sufficient for satisfactory diagnosis. It has already been shown that that a higher degree of sensitivity for detection of SARS CoV-2 infections is reached through the combination of PCR and antibody tests. [1] [2] [3] Thereby, the sensitivity of PCR alone was higher at the early phase of disease, whereas antibody tests alone were more favourable at later time points. Based on its high specificity and sensitivity, PCR-based detection of viral genomes has been proven as a valuable tool to determine SARS-CoV-2 replication in symptomatic, as well as asymptomatic infected persons. The This article is protected by copyright. All rights reserved. For these reasons, there was a call for the development of test systems for specific detection of antibodies directed towards SARS-CoV-2. 4, 5 The primary concept for developing these antibody tests was certainly not to substitute for PCR technology, but rather to complement it. It was suggested to use antibody tests for i) the confirmatory analysis of clinically apparent SARS-CoV-2 infections, ii) the detection of persons that had undergone clinically inapparent SARS-CoV-2 infection, iii) monitoring the success of immunization in the future. The antibody response to SARS-CoV-2 infection seems to evolve after the onset of clinical symptoms 2, 6 and after the beginning of virus replication and shedding. 7 The scientific community is also aware of the necessity to avoid misinterpretations due to the detection of potentially cross-reacting antibodies directed towards seasonal corona viruses in the SARS-CoV-2 antibody test systems. The initial scientific discussion on the interpretation of antibody test results was usually based on a conventional text book view, which assumed that work by other authors showed that this "typical" kinetics seems to be the exception, as will be shown and discussed in more detail in this review. As soon as more data on SARS-CoV-2 serology were available, it was evident that it is much more complex than initially anticipated. 2, 5, 9 For the practically acting expert, these findings were not surprising, as they were not different from the variable antibody responses seen after other viral infections. This critical review therefore analyzes and tries to explain the variability of antibody responses towards SARS-CoV-2 infection measured in n recently published manuscripts and preprints. In addition, a test regimen based on additional avidity determination is proposed, allowing to cope with "atypical" IgM responses. The studies on IgM and IgG responses towards SARS CoV-2 discussed above show a high degree of variability, independent of the methods and the antigens used for detection, confirming that the problem of variability of the immune response is real. The significance of these conclusions is also supported by data obtained is not unlikely that IgM towards SARS CoV-2 might also persist for longer times in rare cases. The This article is protected by copyright. All rights reserved. Especially The determination of avidity starts with the incubation of serum with the test system in duplicate. After a washing step, one of the assays is immediately This article is protected by copyright. All rights reserved. processed to quantification of bound IgG, whereas the parallel assay is first treated with defined concentrations of urea. After a subsequent washing step, this test is processed for quantification of bound IgG. By comparing the results between the urea-treated and the control assay, an avidity index can be determined and used for differentiation between acute, intermediate and past infections. Avidity determination is now used as a valuable analytical method in many diagnostic laboratories. It is one of the key elements for the confirmation or disproval of acute rubella virus infection, particularly during pregnancy. 37, 46 Avidity determination is instrumental to resolve the complex serology of human herpesviruses, such as EBV EBV 25, 51-58 ), CMV 59-62 ), HHV-6 and HHV-7 63 Figure 4 ). As SARS CoV-2 serology is characterized by high variability, and as avidity determination seems to be the key method to resolve analogous problems in other viral systems, we propose to establish and evaluate avidity determination of SARS CoV-2 IgG in the near future. This approach has a predictable potential to improve serodiagnosis of SARS CoV-2 infections. Establishment of SARS CoV-2 avidity testing requires a specific and shown that stored sera from patients, convalescent for SARS CoV-1 infection, were reactive towards MERS-CoV -a virus that had not been in the human population when the sera had been collected. 80 As sera from healthy controls This article is protected by copyright. All rights reserved. from the same time did not react with MERS-CoV, the dependence of cross reactivity on the antibodies originally induced by SARS-CoV-1 was ensured. This cross reactivity can be explained by common epitopes of analogous proteins of related viruses. It remains to be determined, whether this type of rather strong reactivity is caused by high avidity of the involved antibodies. A more complex type of interaction between the viruses and the immune system has been reported by Chan et al. 79 This article is protected by copyright. All rights reserved. It has been established that long-lasting protective humoral immunity requires the generation of high avidity IgG and memory B cells that can generate such antibodies upon adequate stimulation. [81] [82] [83] [84] [85] [86] [87] Therefore, the determination of the kinetics of IgG avidity maturation will be necessary for getting an estimation about the quality as well duration of protection towards reinfection by SARS CoV-2. The present discussion on reinfection with SARS CoV-2 is positioned between the suggestions by Edridge et al. 88 This article is protected by copyright. All rights reserved. In line with these considerations and established work [81] [82] [83] [84] [85] [86] [87] , it is also predictable that avidity determination of IgG established through immunization with SARS CoV-2 antigens in the future will be essential to ensure the quality of protection towards the virus. In line with the findings by Konio et al. 86 , the parallel measurement of antibody concentration and avidity might determine the hopeful success of future vaccination programs towards SARS-CoV-2. The serological response after infection with SARS CoV-2 is highly variable, showing IgM preceding IgG, occurring in parallel with IgG, appearing in a delayed mode compared to IgG, as well as missing IgM after acute infection. In analogy to the findings for many other virus groups and particularly for SARS CoV-1, the establishment avidity determination for SARS CoV-2 infection is proposed to ensure unambiguous serological diagnosis. It is predicted that avidity determination might allow differentiation between cross reacting corona viruses, analysis of reinfections with SARS CoV-2 and help to determine the quality of the immune status after future immunization programs. Group b: Five cases showed IgG, but not IgM on day 0. In three of these cases, the IgM response was detectable on day 5 ("delayed IgM response"). In two cases, no IgM response was detectable at day 5, but acute infection was confirmed by the increase in the IgG response between day 0 and day5. This article is protected by copyright. All rights reserved. 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