Serological anti-SARS-CoV-2 neutralizing antibodies association to live virus neutralizing test titers in COVID-19 paucisymptomatic/symptomatic patients and vaccinated subjects
Antonio CristianoMarzia NuccetelliMassimo PieriSerena SarubbiMartina PelagalliGraziella CalugiFlaminia TomassettiSergio Bernardini
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Antibody titer
Virus quantification
Plaque-forming unit
HSL and HSV
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2019-20 coronavirus outbreak
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Abstract While there are various attempts to administer COVID-19-convalescent plasmas to SARS-CoV-2-infected patients, neither appropriate approach nor clinical utility has been established. We examined the presence and temporal changes of the neutralizing activity of IgG fractions from 43 COVID-19-convalescent plasmas using cell-based assays with multiple endpoints. IgG fractions from 27 cases (62.8%) had significant neutralizing activity and moderately to potently inhibited SARS-CoV-2 infection in cell-based assays; however, no detectable neutralizing activity was found in 16 cases (37.2%). Approximately half of the patients (~ 41%), who had significant neutralizing activity, lost the neutralization activity within ~ 1 month. Despite the rapid decline of neutralizing activity in plasmas, good amounts of SARS-CoV-2-S1-binding antibodies were persistently seen. The longer exposure of COVID-19 patients to greater amounts of SARS-CoV-2 elicits potent immune response to SARS-CoV-2, producing greater neutralization activity and SARS-CoV-2-S1-binding antibody amounts. The dilution of highly-neutralizing plasmas with poorly-neutralizing plasmas relatively readily reduced neutralizing activity. The presence of good amounts of SARS-CoV-2-S1-binding antibodies does not serve as a surrogate ensuring the presence of good neutralizing activity. In selecting good COVID-19-convalescent plasmas, quantification of neutralizing activity in each plasma sample before collection and use is required.
Convalescent plasma
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Discussion and Summary The colorimetric neutralization test for herpes simplex described in this report is considerably easier to conduct than neutralization tests in mice or embryonated eggs, and lends itself to large-scale studies. Also, the constant virusvarying serum technique, widely regarded as more sensitive than the constant serum-varying virus method for detecting differences in levels of neutralizing antibody, is utilized. As a tool for epidemiologic and diagnostic purposes, the neutralization test was found comparable to the complement fixation test in its ability to demonstrate 4-fold or greater titer rises. It would appear that either the complement fixation or colorimetric neutralization test would be adequate for diagnosis of primary herpetic infections, and that little would be gained diagnostically by the use of both tests except in instances where equivocal results were obtained with one test. Only 2 of 31 4-fold or greater rises in antibody titer were detected by the neutralization test, but not the complement fixation test, and only 3 of the 31 antibody rises were detected by the complement fixation test, but not the neutralization test. Although the neutralizing antibody titers tended to be somewhat higher than the complement-fixing antibody titers, they both increased at approximately the same rate in cases of primary herpetic infections. This is in contrast to the antibody response in many other viral diseases in which neutralizing antibodies appear long before complement-fixing antibodies, and may even reach their maximal titer by the time of onset of the illness, e.g., poliomyelitis (19). The neutralizing antibody titers detected by the colorimetric method showed good correlation with the complement-fixing antibody titers for the same sera (r = 0.82). This correlation between complement-fixing and neutralizing antibody titers was noted for individuals in all age groups studied, and it would appear, on the basis of the limited amount of data available, that production of both types of antibody is stimulated by recurrent herpetic infections, or that complement-fixing antibody persists for as long after primary infections as does neutralizing antibody.
Complement fixation test
Antibody titer
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Cytomegalovirus
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ABSTRACT A serum neutralization assay (SN) was compared with the official plaque reduction neutralization test for the quantitation of West Nile virus antibodies. A total of 1,348 samples from equid sera and 38 from human sera were tested by these two methods. Statistically significant differences were not observed, thus supporting the use of SN for routine purposes.
West Nile virus
Virus quantification
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Summary The 7 S and 19 S rabbit antibodies to herpes simplex virus (HSV) from early and late (hyperimmune) sera differed in their ability to sensitize virus for subsequent neutralization by either complement (C′) or anti-γ-globulin (GAR). The early 7 S and 19 S antibodies showed low to negligible neutralizing activity in the absence of C′ or GAR. When C′ was added, however, both of these antibodies showed enhanced neutralizing activity. The early 7 S but not the early 19 S antibody was also capable of sensitizing virus for subsequent neutralization by GAR. The late 19 S antibody could neutralize virus in the absence of C′ or GAR, but its activity was enhanced in the presence of C′ or GAR. The late 7 S antibody showed high neutralizing activity in the absence of C′ or GAR. In the presence of C′, the neutralization rate constants (K) but not the neutralization titers of the late 7 S antibody were enhanced. In contrast, the neutralization titers of the late 7 S antibody were enhanced approximately threefold with GAR. The neutralizing activity of the early and late 19 S antibodies with C′ or GAR was sensitive to inactivation by 2-ME. Similarly, the neutralizing activity with C′ of the early 7 S antibody and the enhanced rate of neutralization with C′ of the late 7 S antibody were sensitive to inactivation by 2-ME. In contrast, 2-ME did not reduce the neutralization titers of the early and late 7 S antibodies in the presence of GAR.
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Among several infections in mice, both lethal and nonlethal, it was found that an antiglobulin plaque neutralization test was superior to a standard plaque neutralization test for the detection of antibody. In most cases, the antiglobulin plaque neutralization test detected antibody sooner and to higher titer than the standard plaque neutralization test. In one of the nonlethal infections, the antiglobulin plaque neutralization test detected antibody concentrations ranging from 1:80 and 1:1280 where it was otherwise undetectable by a standard plaque neutralization test, complement-fixation test or hemagglutination-inhibition test. In certain hyperimmune sera there was sometimes little difference in the results of the two tests, but absorption of the antisera with viruses revealed significantly higher titers by the antiglobulin plaque neutralization test than the standard plaque neutralization test in the remaining antibody population in at least 2 of 4 sera examined. In addition, infectious virus-antibody complexes which remained after absorption of antibody could be neutralized by the addition of antimouse gamma globulin but not by control fluids.
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The production of neutralizing antibodies (NAbs) is a correlate of protection for many human vaccines, including currently licensed vaccines against flaviviruses. NAbs are typically measured using a plaque reduction neutralization test (PRNT). Despite its extensive use, parameters that impact the performance of the PRNT have not been investigated from a mechanistic perspective. The results of a recent phase IIb clinical trial of a tetravalent dengue virus (DENV) vaccine suggest that NAbs, as measured using a PRNT performed with Vero cells, do not correlate with protection. This surprising finding highlights the importance of understanding how well the PRNT captures the complexity of the NAb response to DENV. In this study, we demonstrated that the structural heterogeneity of flaviviruses arising from inefficient virion maturation impacts the results of neutralization assays in a cell type-dependent manner. Neutralization titers of several monoclonal antibodies were significantly reduced when assayed on Vero cells compared to Raji cells expressing DC-SIGNR. This pattern can be explained by differences in the efficiency with which partially mature flaviviruses attach to each cell type, rather than a differential capacity of antibody to block infection. Vero cells are poorly permissive to the fraction of virions that are most sensitive to neutralization. Analysis of sera from recipients of live-attenuated monovalent DENV vaccine candidates revealed a strong correlation between the sensitivity of serum antibodies to the maturation state of DENV and cell type-dependent patterns of neutralization. Cross-reactive patterns of neutralization may be underrepresented by the "gold-standard" PRNT that employs Vero cells.Cell type-dependent patterns of neutralization describe a differential capacity of antibodies to inhibit virus infection when assayed on multiple cellular substrates. In this study, we established a link between antibodies that neutralize infection in a cell type-dependent fashion and those sensitive to the maturation state of the flavivirus virion. We demonstrated that cell type-dependent neutralization reflects a differential capacity to measure neutralization of viruses that are incompletely mature. Partially mature virions that most efficiently bind maturation state-sensitive antibodies are poorly represented by assays typically used in support of flavivirus vaccine development. The selection of cellular substrate for neutralization assays may significantly impact evaluation of the neutralization potency of the polyclonal response. These data suggest that current assays do not adequately capture the full complexity of the neutralizing antibody response and may hinder the identification of correlates of protection following flavivirus vaccination.
Vero cell
Dengue vaccine
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Abstract Background There is a crucial need for effective therapies that are immediately available to counteract COVID-19 disease. Recently, ELISA binding cross-reactivity against components of human epidemic coronaviruses with currently available intravenous immunoglobulins (IVIG) Gamunex-C and Flebogamma DIF (5% and 10%) have been reported. In this study, the same products were tested for neutralization activity against SARS-CoV-2, SARS-CoV and MERS-CoV and their potential as an antiviral therapy. Methods The neutralization capacity of six selected lots of IVIG was assessed against SARS-CoV-2 (two different isolates), SARS-CoV and MERS-CoV in cell cultures. Infectivity neutralization was measured by determining the percent reduction in plaque-forming units (PFU) and by cytopathic effects for two IVIG lots in one of the SARS-CoV-2 isolates. Neutralization was quantified using the plaque reduction neutralization test 50 (PRNT 50 ) in the PFU assay and the half maximal inhibitory concentration (IC 50 ) in the cytopathic/cytotoxic method (calculated as the minus log 10 dilution which reduced the viral titer by 50%). Results All IVIG preparations showed neutralization of both SARS-CoV-2 isolates, ranging from 79 to 89.5% with PRNT 50 titers from 4.5 to >5 for the PFU method and ranging from 47.0%-64.7% with an IC 50 ~1 for the cytopathic method. All IVIG lots produced neutralization of SARS-CoV ranging from 39.5 to 55.1 % and PRNT 50 values ranging from 2.0 to 3.3. No IVIG preparation showed significant neutralizing activity against MERS-CoV. Conclusion In cell culture neutralization assays, the tested IVIG products contain antibodies with significant cross-neutralization capacity against SARS-CoV-2 and SARS-CoV. However, no neutralization capacity was demonstrated against MERS-CoV. These preparations are currently available and may be immediately useful for COVID-19 management.
Cytopathic effect
Infectivity
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