Biotechnology Test Pdf

[Trisha Quercioli]

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We then tested different animal sera in the sVNT assays to demonstrate species-independent performance. Results from mice and rabbits immunized with the SARS-CoV-2 RBD protein demonstrate very potent neutralizing activity in the SARS-CoV-2 sVNT (Fig. 3a). Similarly, sera from ferrets infected with SARS-CoV and rabbits immunized with inactivated SARS-CoV also display an efficient dose-dependent inhibition of the interaction between hACE2 and the SARS-CoV RBD in the SARS-CoV sVNT (Fig. 3b).

To demonstrate specificity, we tested different panels and confirmed that the SARS-CoV-2 sVNT can differentiate antibody responses to SARS-CoV-2 from those to other human CoV infections (Fig. 3c). For human sera from patients with 229/NL63 or OC43 infection and alpaca sera from experimental MERS-CoV infection, there is no detectable cross-neutralization. For SARS sera, there is some level of cross-reactivity, not unexpected given their close genetic relatedness and what was reported previously3,7. When analyzed by the SARS-CoV and SARS-CoV-2 sVNT assays side-by-side, neutralizing sera from patients who had SARS could be differentiated from sera from patients who had COVID-19 (Fig. 3d,e).

During the investigation of potential cross-reactivity between SARS sera and SARS-CoV-2 virus, we made several notable observations. First, despite the lack of cross-neutralization by SARS sera against the live SARS-CoV-2 virus in cVNT observed by us and other groups13,14, we detected some level of cross-neutralization in sVNT (Fig. 3c), indicating that sVNT is more sensitive than cVNT. Second, SARS NAbs are detectable for at least 17 years in recovered patients (Fig. 3c,e). Third, the cross-neutralization level is higher in SARS sera sampled in 2020 than in those sampled in 2003 (Fig. 3c), although the homologous neutralizing level of the 2020 sera (Fig. 3e) is lower than that of the 2003 sera (Fig. 3d); this is also confirmed by determining RBD-binding antibodies using an indirect ELISA assay (Extended Data Fig. 3). Finally, we have found that the N-specific antibody level is much lower in the 2020 SARS sera than in the 2003 samples (Fig. 3f).

Although many COVID-19 laboratory-based or point-of-care antibody test kits are commercially available, none is capable of measuring NAbs. The cVNT and pVNT platforms remain the only platforms for detection of NAbs. However, both require live viruses and cells, highly skilled operators, and days to obtain results. They are thus not suitable for mass production and testing on a commercial scale, even in the most developed nations.

The data presented here demonstrated that sVNT is as specific as, but more sensitive than, cVNT in the cell types tested here (Fig. 4). In our initial optimization studies, we found that the RBD protein performed better than the S1 protein (Extended Data Fig. 1). We have also compared the RBD proteins produced in insect and mammalian cells and found very similar performance (Extended Data Fig. 5). It is still possible to further improve the sensitivity of the sVNT platform in future by protein engineering on either the RBD- or the ACE2-binding interface. The mAb studies presented in Extended Data Fig. 2 demonstrate that the RBD sVNT measures genuine NAbs, whereas the RBD ELISA is unable to differentiate between BAbs and NAbs. It can therefore be concluded that the RBD-based sVNT is a robust assay platform for reliable quantification of RBD-targeting NAbs. It should be noted that not all NAbs are necessarily RBD-binding antibodies, as indicated by past studies with SARS-CoV that show antibodies to other regions in the S1 or S2 protein can also play a role in virus neutralization18. However, studies based on both SARS-CoV and SARS-CoV-2 suggest that the RBD-targeting NAbs are immunodominant during both SARS and COVID-19 infections19,20. In our study, we used 60 patient serum samples of varying NAb levels, and the 3-way correlation studies presented in Fig. 4 clearly demonstrate that the correlation between sVNT and cVNT is as good as, if not better than, that between pVNT and cVNT. This indicates that non-RBD-targeting antibodies, which could be measured in pVNT, but not in sVNT, are unlikely to play a major role in SARS-CoV-2 neutralization, consistent with previous findings19,20,21.

The major advantage of sVNT is that it can be rapidly conducted in most research or clinical laboratories without the need to use live biological materials and biosafety containment. The sVNT is also amenable to high-throughput testing and/or fully automated testing after minimal adaptation.

In addition, sVNT offers a key advantage over most ELISA or point-of-care tests in its ability to detect total NAbs in an isotype-independent manner. This will not only simplify the testing strategy but also further increase the test sensitivity. As shown in Fig. 2b for the serum panel of patients with COVID-19 showing low IgM and IgG in the isotype-specific ELISAs, the sVNT assay still detected a substantial level of NAbs. Although the mechanism needs further investigation, there are at least two possibilities: the presence of other immunoglobulin isotypes or neutralization synergy (cooperativity from the combination of different isotype antibodies targeting different neutralization-critical epitopes), as previously observed for HIV and other viruses28,29,30. Our preliminary analyses indicate that neutralization synergy is more likely the mechanism. First, from the human mAb study, we have found some evidence of synergy between two neutralizing mAbs, AR6949 and AR6959 (Extended Data Fig. 2i). Second, IgA testing indicates that there was no high level of RBD-specific IgA in the low IgM/low IgG group (Extended Data Fig. 6).

The results obtained from the two SARS serum panels of 17 years apart are notable. The presence of long-lasting NAbs 17 years after the initial infection is encouraging news for patients who have recovered from COVID-19, considering the close relationship between the two viruses. The mechanism and biological significance of the increased cross-neutralization towards SARS-CoV-2 coupled with the decrease/disappearance of N-specific antibodies 17 years after infection warrant further investigation in the context of better understanding SARSr-CoV immune response dynamics.

In summary, we have addressed the challenge of COVID-19 serology with an approach that enables the detection of NAbs in an easy, safe and rapid manner with enhanced specificity and sensitivity. Although the sVNT assay may never be able to completely replace cVNT, our data indicate that the performance of sVNT is well correlated with that of both cVNT and pVNT. Its application can cover many aspects of COVID-19 investigation from contact tracing, seroprevalence surveying and reservoir/intermediate animal tracking to the assessment of herd immunity and longevity of protective immunity. It can also be used to assess vaccine efficacy during preclinical and clinical trials of different vaccine candidates and to monitor neutralizing titers in vaccinees after mass vaccination in human populations.

In Singapore, the sera from patients with COVID-19 used in this study were from the Singapore PROTECT study as described previously31. Sera from patients who had recovered from SARS from 2003 were as previously described32. For SARS recall sampling in 2020, we contacted and obtained blood from consenting individuals previously admitted for SARS (ethics approval number: NHG DSRB E 2020/00091). The human CoV serum panel included post-infection samples from subjects confirmed positive for CoV 229/NL63 and CoV OC43 using the SeeGene RV12 respiratory multiplex kit in a previous study (ethics approval number: NUS-IRB 11-3640)32. Negative control sera were obtained from residual serum samples from previous unrelated studies. In Nanjing, China, sera from convalescent patients with COVID-19 were collected with written informed consent and approved by the ethics committee of The Second Hospital of Nanjing (ethics approval number: 2020-LS-ky003). Mouse and rabbit anti-SARS-CoV-2 RBD sera and monoclonal antibodies raised against the SARS-CoV-2 RBD were all from GenScript. Rabbit and ferret anti-SARS-CoV sera and alpaca anti-MERS-CoV sera were as described in previous studies33,34.

Statistical analysis was performed using GraphPad Prism 7 software. The differences between negative control and COVID-19 test sera were analyzed using an unpaired t-test. The differences between paired SARS serum in SARS-CoV-2 sVNT and SARS-CoV sVNT were analyzed using a paired t-test. Correlations between sVNT and cVNT or pVNT were analyzed using Pearson correlation coefficients. All data presented are derived from two independent experiments.

We thank S. Tang, Y. Shen, N. Mao, W. Shao and L. Zhu for technical assistance, advice and logistics support with assay development and testing; Y. Peng, A. Gamage, B. L. Lim and X. M. Ong for assistance with protein purification, sample management and testing; Y. Abdad and L. W. L. Tan for help with human CoV serum collection; V. Vijayan, B. Ng and V. Sivalingam of the Duke-NUS Medical School ABSL3 facility for logistics management and assistance. L.-F.W. and D.E.A. are supported by grants from the Singapore National Research Foundation (NRF2016NRF-NSFC002-013) and the National Medical Research Council (STPRG-FY19-001 and COVID19RF-003).

L.-F.W. conceived and guided the study. C.W.T., W.N.C., X.Q., P.L., C.T., V.C.-W.C., W.R.S., R.F. and D.E.A. performed laboratory work including data analysis. M.I.-C.C., Z.H., B.E.Y., Y.-J.T., Y.Y. and D.C.L. provided necessary samples and coordination for the study. L.-F.W. initiated the manuscript writing with input from all authors.

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