However, how neutralizing antibodies inhibit infection in vitro remains unclear. The neutralizing anti-VLP antisera were able to inhibit virus binding to target cells efficiently. In addition, post-attachment treatment of virus-bound cells with the anti-VLP antisera also neutralized virus infection, although the antibody concentration required was higher than that of the pre-attachment treatment. The standard error s. This is, in most cases, the ratio of two values directly reported in the immunogenicity studies.
However, this approach does not account for situations in which the neutralization assay had neutralization titers below the LOD, therefore we also used a second method, in which we estimated this value by extracting the neutralization titers from the figures in each immunogenicity study Supplementary Table 1 and computing the mean neutralization titer for vaccinated and convalescent individuals in each study using censoring regression equation 1.
Additionally, although it was in principle possible to compute the standard deviation of neutralization levels for each study as above , these appeared somewhat confounded by the varying numbers of individuals between studies, hence we fitted the above model using 1 the standard deviation estimates for each study, 2 one standard deviation from one larger study to which we had direct access to raw data 3 that is, no manual data extraction required , and 3 an estimate of the standard deviation for all studies pooled together.
The two different methods of estimating the mean neutralization level for each study and the three methods of estimating the standard deviation of the neutralization levels give rise to six versions of the above model.
All of these versions of the model were fitted, and the estimated protective levels were very similar Extended Data Fig. The above modeling approach assumed that neutralization levels were normally distributed. Here, we present a method for determining a protective threshold that is free of assumptions regarding the distribution of neutralization levels.
This model assumes that there is a protective neutralization level, T , above which individuals will be protected from infection and below which individuals will be susceptible. The protective efficacies observed in phase 3 clinical trials of vaccinated individuals and another large cohort study of convalescent individuals 1 ; Supplementary Table 2 are denoted by E s.
These represent the proportion of individuals in each study, s , who should possess a neutralization level above the protective threshold. It follows then that the number of individuals above the protective threshold in study s is a function of T , which we denote K s T. Therefore, the probability of observing K s T individuals above the protective threshold, given that there were N s individuals in the immunogenicity study which are much smaller than the phase 3 studies , is given by.
To determine one protective threshold using the results of all efficacy studies in this paper, we construct a likelihood function. Note that this likelihood function is discontinuous as the threshold T is varied. Therefore, we evaluate this likelihood function with the threshold T set equal to all observed neutralization levels n i across all studies, and find the value of T that maximizes this likelihood Extended Data Fig.
This method determines a protective level at which the proportion of individuals with neutralization levels above the threshold is in greatest agreement with the observed protective efficacy of that vaccine.
Equation 8 is the likelihood function that should be adopted when neutralization measurements are not affected by an LOD. In the case that some neutralization levels are below the LOD, the likelihood function is adjusted as follows:. C s is the number of censored values in study s and Q is the cumulative binomial distribution function.
This later term considers the probability that as many as all of the censored values were below the threshold T given the protective efficacy of the study E s. Resampling was performed so as to preserve the total number of neutralization levels in each study. These randomly generated samples of the original data were then fitted in the same way as described above, which generated 1, corresponding estimates of the protective neutralization level.
To determine the ability of the model to predict the efficacy of a vaccine, we performed a leave-one-out analysis in which we systematically excluded one of the vaccine studies or the convalescent study and performed the same model-fitting procedure described above. Using the model fitted on the subset of the studies we then estimated the efficacy of the vaccine that was left out from the fitted model.
This leave-one-out analysis was performed for all versions of the logistic model that is, using the six methods of estimating the mean neutralization level and standard deviation outlined above. The predicted efficacy for each vaccine and convalescence obtained while leaving the study out are plotted against the reported efficacy in Fig. In Fig. That is, these represent. We also tested if the protective neutralization level was different between mild and severe infection by fitting the combined dataset with two different mathematical models.
A number of studies have analyzed the decay in neutralization titer in convalescent subjects. These studies have generally shown a rapid early decay that slows with time 3 , 4 , 5 , 59 , We identified one study by Widge et al. Note that this restricted convalescent time course was used only so that we could compare decay in vaccination and convalescence over a similar time course time-limited by the vaccination data. This is the estimate that was used in the predictive model presented in Figs.
The decay in efficacy with time Fig. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Lumley, S. Kim, J. Wheatley, A. Gaebler, C. Nature , — Dan, J. Science , eabf Wang, P. Hobson, D. The role of serum haemagglutination-inhibiting antibody in protection against challenge infection with influenza A2 and B viruses. Coudeville, L. Relationship between haemagglutination-inhibiting antibody titres and clinical protection against influenza: development and application of a bayesian random-effects model.
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Ella, R. Safety and immunogenicity of an inactivated SARS-CoV-2 vaccine, BBV interim results from a double-blind, randomised, multicentre, phase 2 trial, and 3-month follow-up of a double-blind, randomised phase 1 trial. Widge, A. Rodda, L. Cell , — Pradenas, E. Med NY 2 , — Chia, W. Chen, R. Wang, Z. Wu, K. Serum neutralizing activity elicited by mRNA vaccine. Liu, Y. Neutralizing activity of BNTb2-elicited serum. Zhou, D. Sette, A. Rydyznski Moderbacher, C. Juno, J. Cromer, D.
Amanna, I. Duration of humoral immunity to common viral and vaccine antigens. Antia, A. Heterogeneity and longevity of antibody memory to viruses and vaccines. PLoS Biol. Monto, A. The second dose was administered 21 days after the first one. All volunteers had blood drawn within 2 days before the first vaccine dose and additional blood samples were then collected after 14, 21, 28, 42, and 56 days.
Additionally, samples collected before January were assessed to evaluate the clinical specificity of the sVNT.
Frozen samples were thawed for 1 h at room temperature on the day of the analysis. Re-thawed samples were vortexed before the analysis. The study protocol was in accordance with the Declaration of Helsinki.
All vaccinated participants provided informed consent prior to the collection of data and specimens EudraCT registration number: Neutralizing capacity was estimated by performing an sVNT. First, NAbs if present react with the RBD antigen coated on paramagnetic microparticles to form a complex. Second, the acridinium-ester-labeled ACE2 conjugate is added to competitively bind to the RBD-coated particles, which have not been neutralized by the NAbs if present from the sample, and these form another reaction mixture.
Under a magnetic field, magnetic particles are adsorbed to the wall of the reaction tube, and unbound materials are washed away by the wash buffer. The resulting chemiluminescent reaction is measured in relative light units RLUs , with an inverse relationship between the amount of NAbs and the RLU value detected.
According to the manufacturer, it shows excellent positive Shenzhen, China Internal quality controls negative and positive and 6 sera from COVID patients at various NAbs titers were analyzed 10 times in a row to calculate the within-run precision of the assay. The positive internal quality control was also analyzed for a period of 15 days to calculate the between-run precision. A total of 71 random samples i. Details about the method are presented in Supplementary Materials. The Roche NCP total assay was also used to determine the serological status of vaccinated participants before vaccine injection.
Descriptive statistics were used to analyze the data. Specificity was defined as the proportion of pre-pandemic samples classified as negative.
A Mann—Whitney test was used to assess potential differences in median time since diagnosis in mild versus moderate—severe COVID patients.
A simple linear regression and Pearson correlations were computed to assess the potential association between NAb titers and antibody titers obtained using 6 non-neutralizing commercial methods. Inter-rater agreements were also determined. NAb titers among the two vaccinated groups at different time points were tested using an ANOVA multiple comparisons test. The mean of the NAb titers was 3. The within-run CV ranged from 4.
A higher CV was observed using the negative quality control The between-run CV using the positive internal quality control was Precision of the sVNT using controls and patient samples. All materials were analyzed 10 times in a row. These were the only two discordant results out of 71 samples, and they were close to the positivity cut-off of the sVNT i.
The mean NAb titer in moderate—severe patients was significantly higher compared to mild patients versus With the exception of the Roche S total and Ortho IgG assays, higher correlations were obtained for IgG assays and weaker correlations for total assays Figure 3.
The agreement between methods was good and ranged from Black dotted lines correspond to the positivity threshold of each assay.
The Figure 4 represents the evolution of NAbs in a group of 90 vaccinated individuals. At day 14, the rate of seroconversion after the first dose was Seven days after the administration of the second dose, a The evolution of NAbs in a group of 90 vaccinated participants.
Uninfected individuals are represented in yellow and previously infected individuals are represented in green turquoise. At day 14, a significant Considering each time point separately, NAbs were always statistically higher in previously infected individuals compared to uninfected individuals Figure 4. NAb titers obtained in the first group moderate—severe and mild COVID , compared to those obtained in the group of vaccinated participants, at day In this study, we evaluated the neutralizing capacity in two groups of COVID patients and healthcare professionals who had received the complete dose regimen of the BNTb2 vaccine.
For that purpose, an sVNT was used. An excellent agreement of We also found that the specificity of the sVNT using a panel of pre-pandemic samples was excellent i. A potential cut-off refinement using a ROC curve analysis did not reveal the usefulness of an optimized cut-off, as already performed for some serological assays [ 16 , 17 , 18 , 19 , 20 , 21 , 22 ]. The excellent specificity observed in our study was in line with that claimed by the manufacturer i.
The precision of the assay was also good Table 2. As observed in previous reports [ 23 ], a stronger neutralizing activity was identified in moderate—severe compared to mild COVID patients Figure 1. The slow decay in NAbs with time was also consistent with some reports [ 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ], especially considering mild—moderate patients.
Compared to SARS-CoV-2 antibody assays, only neutralization activity assays reliably measure the real protective immunity of generated antibodies. There is also a high demand for the neutralization tests in specific clinical and industrial settings e. However, the conventional virus neutralization test requires live pathogens and is reserved for very specialized laboratories, requiring a high workload, skillful operators, specific and expensive facilities, and a biosafety level 3 laboratory, and on top of that, they have a low result throughput [ 8 , 9 ].
The use of automated and quantitative assays with a short turn-around time that have a well-documented correlation with the neutralizing activity should be preferred [ 7 , 9 , 31 ]. This is in line with the findings of Legros et al. Patel et al. We therefore confirm that the strongest correlations are observed using anti-S or anti-RBD assays [ 5 , 29 , 34 , 35 , 36 ] and our study highlights that correlations were especially high with the IgG assay.
The fact that anti-NCP assays had a low correlation with the neutralizing activity was expected, as NAbs are directed against the S protein [ 37 ]. Nevertheless, it is important to keep in mind that a few patients may develop specific antibodies, i. We therefore think that the assessment of the neutralizing activity using an sVNT on an automated platform without the disagreement of the gold standard technique might be valuable.
NAbs were measured at baseline, i. So far, few reports have investigated the neutralizing response in vaccinated subjects [ 41 , 42 , 43 , 44 , 45 , 46 ] and they mainly included few participants, only investigating the effect of the first dose [ 42 , 43 , 45 ], or did not include previously infected individuals [ 46 ]. In our study, a significant increase in NAb titers was seen after the first dose i. Interestingly, the neutralizing capacity was similar when comparing previously infected individuals at baseline and naive individuals after the first dose, an observation that is similar to that of Manisty et al.
After the second dose, a significant increase in NAb titers was only observed in uninfected individuals i. Afterwards, the peak of the neutralizing capacity seems to have been reached at day 42 i. Terpos et al. All participants were considered positive 7 days after the second dose.
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