Vaccine monitoring shows that focused immunization with SARS-CoV-2 receptor-binding domain 1 provides a better neutralizing antibody response than full-length spike protein 2

Effective tools to monitor SARS-CoV-2 transmission and humoral immune responses are highly needed. 17 Protective humoral immunity involves neutralizing antibodies and will be a hallmark for the evaluation 18 of a vaccine response efficacy. Here we present a sensitive, fast and simple neutralization ELISA method 19 to determine the levels of antibody-mediated virus neutralization. We can show that it is strongly 20 correlated with the more elaborate plaque reduction neutralization test (PRNT) ( ρ = 0.9231, p < 0.0001). 21 Furthermore, we present pre-clinical vaccine models using recombinant receptor binding domain (RBD) 22 and full-length spike

Introduction 33 34 COVID-19 has within a short time become a worldwide health crisis and the scientific community has 35 stepped up in earnest to this unprecedented challenge to develop diagnostic and therapeutic tools to 36 contain and treat the pandemic. As of September 2020, there were 231 vaccine candidates in the pipeline 37 and more than 30 in clinical trials 1 . Apart from vaccines to prevent SARS-CoV-2 infection, passive anti-38 SARS-CoV-2 antibody therapy to treat COVID-19 patients has emerged as a treatment possibility 2 . 39 Studies have reported that the majority of COVID-19 patients develop neutralizing antibodies targeting 40 the spike glycoprotein within the first two weeks after symptom onset 3-7 , and that SARS-CoV-2-derived 41 antibodies have a protective effect in COVID-19 animal models such as rhesus macaques 8 and rodents 9-

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The current standard method to evaluate the presence of neutralizing antibodies in the blood is the plaque 49 reduction neutralization test (PRNT). While it remains the gold standard due to its specificity and 50 sensitivity 15-18 , the PRNT is labour-and time-intensive, difficult to standardize, and requires highly 51 specialized personnel in high biosafety levels laboratories. Thus, it is of critical importance to develop 52 reliable and convenient methods to assess the virus-neutralizing capacity of patient-or animal-derived 53 antibodies to select convalescent plasma donors, develop mAbs-based therapeutics, and evaluate the 54 efficacy of vaccination strategies.

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Here we describe the development of a quick, sensitive, and easy-to-operate neutralization ELISA-based 57 test for the determination of neutralizing antibodies based on the interaction between recombinant human 58 ACE-2 ectodomain and the SARS-CoV-2 RBD. We benchmarked our assay with the PRNT and two 59 commercially available tests. Using a previously described cohort of PCR-confirmed COVID-19 60 convalescent patients 19 , we measured the neutralization potency and the relative titers of IgG, IgM, and 61 IgA against the RBD, spike, and protein N. Furthermore, we evaluated the vaccine responses in pre-   GeneArt (Thermo Fisher Scientific) and subcloned into a pcDNA3.4 expression vector. The production 94 and purification of SARS-CoV-2 protein N, RBD, and trimeric prefusion-stabilized spike protein 95 ectodomain used for immunization 19

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Unless otherwise stated, all incubation steps were performed for 1 h at RT in a shaking platform, and the 175 plates were washed between steps with PBS-T. Plates were developed with TMB One for 7 min for IgG, 176 and 10 min for IgM and IgA, and the reaction was stopped with 0.3 M H2SO4, and the OD measured as 177 described previously. Antibody titers against RBD have previously been reported by our group 19 . four-parameter non-linear curve fitting and reported as AU/ml as described elsewhere 19 . P values < 0.05 247 were considered statistically significant. 251 We synthesized recombinant human ACE-2 ectodomain (aa 17-740) and SARS-CoV-2 RBD (aa The intra-and inter-assay CV were found to be satisfactory (4.21% and 12.95%, respectively).

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Altogether, these results demonstrate that the ELISA-based neutralization test is robust, time- Assay validation 295 We compared the performance of the developed antibody neutralization ELISA to an authentic

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In parallel, we measured the titers of IgG, IgM, and IgA against RBD, spike, and protein N using 319 a direct ELISA approach (Figure 4), which was published recently 19 . The neutralization potency,    repertoire is highly distributed on the spike protein surface and that a focused immunization 442 approach using only the RBD might be relevant to consider in future vaccine development 443 strategies. Compatible with such notion is our observation that sera from convalescent 444 individuals that have been exposed to the whole virus had several hundredfold less neutralizing 445 capacity than the mice immunized with RBD. However, some caution should be taken since we 446 do not know whether the mice response can be directly translated to the human situation.

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Nevertheless, the results suggest that the RBD without any carrier or other specific formulations 448 could be an excellent vaccine candidate.

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To further address whether the antibody neutralization ELISA could be used to monitor scale. Moreover, our data suggest that using RBD as an immunogen compared with full-length 468 spike protein might be a better strategy in creating a robust neutralization antibody response.

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This should be considered when developing next-generation vaccines against SARS-CoV-2 or 470 related escape strains that could potentially emerge after the present pandemic.