key: cord-0957382-4nozg8uc
authors: Alvim, R. G. F.; Lima, T. M.; Rodrigues, D. A. S.; Marsili, F. F.; Bozza, V. B. T.; Higa, L. M.; Monteiro, F. L.; Leitao, I. C.; Carvalho, R. S.; Galliez, R. M.; Castineiras, T. M. P. P.; Nobrega, A.; Travassos, L. H.; Ferreira, O. C.; Tanuri, A.; Vale, A. M.; Castilho, L. R.
title: AN AFFORDABLE ANTI-SARS-COV-2 SPIKE ELISA TEST FOR EARLY DETECTION OF IgG SEROCONVERSION SUITED FOR LARGE-SCALE SURVEILLANCE STUDIES IN LOW-INCOME COUNTRIES
date: 2020-07-15
journal: nan
DOI: 10.1101/2020.07.13.20152884
sha: a601d4824a571e9ab1787a04eea7c9ee7c720677
doc_id: 957382
cord_uid: 4nozg8uc

We describe a cost-effective, scalable technology to produce SARS-COV-2 spike (S) protein based on stable expression in HEK293 cells, and its use to develop a highly specific and sensitive ELISA test. The assay allows early detection of anti-S IgG seroconversion and endpoint titers correlate with virus neutralization. The low-cost S-antigen production, together with sample collection by finger prick and dried blood spots, allowed the development of a half-dollar test that fits the urgent need for large-scale serological surveillance in low-income countries.

Tools to monitor and control COVID-19 are urgently needed in the context of the international public health emergency posed by SARS-COV-2 coronavirus pandemics. High-quality recombinant viral antigens are an essential tool to enable the development of serological assays needed for seroprevalence and epidemiological surveillance studies, contact mapping, identification of asymptomatic infections, evaluation of potential vaccine candidates and fundamental research on coronavirus immunity. The structural spike (S) protein of SARS-COV-2 contains the receptor binding domain in its S1 subunit and is also responsible for fusion to the cell membrane through its S2 sub-unit. S protein is known to be a major target for neutralizing antibodies, thus making it a key antigen for the development of sensitive diagnostic tools. Enzyme linked immunosorbent (ELISA) assays based on the S protein have been developed, showing minimal cross-reactivity with sera against commonly circulating coronaviruses [1] and providing a good correlation to neutralizing activity [2] . Here, we focused on establishing the hallmarks for a low-cost ELISA assay based on S protein immunoreactivity, which can be broadly employed in epidemiological surveillance studies in low-income countries. Without compromising the performance of the assay, costs were cut mainly by optimizing antigen production and adapting sample collection and processing, resulting in a total cost estimated in less than half a dollar per sample.

For recombinant production and straightforward purification of the heavily glycosylated S protein, mammalian cell culture in serum-free media is the option of choice. We expressed the soluble ectodomain of the spike (S) protein of SARS-COV-2 in a stabilized prefusion conformation [3] in serum-free, suspension-adapted HEK293-3F6 cells. Differently from previous works that use transient protein expression techniques [2, 3] , we focused on stable, constitutive gene expression, since transgene integration in the cell genome enhances scalability and significantly decreases costs of recombinant protein production in mammalian cells [4] . Due to the challenges posed by the pandemics, such as the urgency and the disruption of international supply chains for reagents and synthetic gene constructs, we decreased time and costs by using an old-fashioned technique of co-transfecting the plasmid containing the S gene and an intellectual property-free plasmid containing a selection marker. A stable recombinant HEK293 cell line showing higher expression than transiently transfected HEK293 or CHO-K1 cells was generated and banked within . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 15, 2020. . https://doi.org/10.1101/2020.07.13.20152884 doi: medRxiv preprint proven for up to 100 days post-transfection (Fig. 1a) , thus making it feasible to develop less costly, long-lasting batch-refeed or perfusion technologies for cell culture. In order to decrease cell cultivation costs and logistics burden, two culture media available as powdered media were evaluated in shake flasks and stirred-tank bioreactors at 300-mL and 1.5-L scale, respectively, and the less expensive HEK-GM medium was able to provide robust cell growth and efficient recombinant protein production ( Figure 1b ). Carrying out cell culture in fed-batch mode by adding pulses of a concentrated nutrient solution over cell cultivation time avoided nutrient depletion and significantly increased viable cell density and secreted S protein levels ( Figure   1b ). Protein isolation from cell culture supernatant was investigated by ultrafiltration/diafiltration (UF/DF) and affinity chromatography (AC) techniques. In spite of the large S protein size and the use of a 100-kDa cut-off membrane device, UF/DF was not able to remove smaller contaminating proteins, and an AC resin bearing an streptavidin mutein ligand was used to allow obtaining the protein in high purity ( Figure 1c ). The affinity resin has shown a relatively high dynamic binding capacity for the S protein (Figure 1d ), and has so far been used for 30+ adsorption/elution/regeneration cycles with no detectable decrease in performance, reducing its impact on the final costs of the purified protein.

An ELISA assay was then established for the detection of anti-S antibodies in serum, plasma and whole blood eluates (S-UFRJ ELISA). A comparison of the UF/DF concentrated S protein to the high-purity AC sample showed that assay performance can be greatly improved by the use of the highly purified antigen (Fig. 1e ). Using serum samples from negative controls and COVID-19 PCR-confirmed patients (see methods), we established that 150 ng of high-purity S protein per well enables clear discrimination of samples (Fig. 1f ). is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 15, 2020. . https://doi.org/10.1101/2020.07.13.20152884 doi: medRxiv preprint (post-COVID-19, n=20). The assay exhibited high specificity as 122 out of 124 of pre-COVID-19 controls scored as negative, below the cut-off (98.39 % specificity) (Fig   2a) . Considering all samples from PCR+ individuals, regardless of the time of collection, 53 out of 66 samples (80.30 % sensitivity) were IgG positive. For comparison, all samples were also tested for anti-SARS-COV-2 IgG using an imported rapid diagnostic test (RDT) that has been approved by the Brazilian regulatory agency (ANVISA) for commercialization in the country. Using this commercial RDT, only 30 out of 66 samples were positive for IgG (45.45% sensitivity) (Fig 2b) .

In order to gain insight into the samples from symptomatic PCR-positive individuals that scored negative for anti-S IgG in the S-UFRJ ELISA assay, samples were grouped as IgM-and IgM+ according to the IgM rapid test. We found that most samples that scored negative for anti-S IgG in our ELISA assay, also scored as negative for IgM in the rapid test (Fig 2c) . On the other hand, the majority of samples that were IgG positive the in S-UFRJ ELISA were also positive for IgM in the rapid test (Fig 2c) . This result suggests that samples from PCR-positive individuals that scored negative for anti-S IgG in our ELISA assay, may have been collected in the very beginning of the disease, only a few days after symptoms onset (DASO), and for that reason scored negative both for IgM in the rapid test and for IgG in our Sbased ELISA. Indeed, when the results of S-UFRJ ELISA were charted against DASO, this was found to be the case: samples were increasingly scored as positive for anti-S IgG according to the time point they had been collected after symptoms onset (Fig 2d) . Accordingly, PCR-positive individuals that have been sampled on different DASO and scored negative in their first sampling, converted to seropositive for anti-S IgG afterwards (data not shown). Anti-S IgG seroconversion rate, detected by S-UFRJ ELISA, increases progressively from 41.66 % to 100 % as a function of DASO, being above 90 % already from day 10 after symptoms onset on (fig 2e) . Of note, S-UFRJ ELISA detects anti-S IgG seroconversion much earlier than the rapid test used here for comparison, which reached a maximum positive rate as low as 71.43% even for DASO of 20 days and above. We further tested serum samples of COVID-19 convalescent patients for their SARS-COV-2 in vitro neutralization ability as measured by PRNT assay. Importantly, analogous to what has been shown by Amanat et al. [2] , we found that sera displaying high anti-S IgG ELISA titers (Fig 2f) . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 15, 2020. . https://doi.org/10.1101/2020.07.13.20152884 doi: medRxiv preprint also presented high neutralization titers (Fig 2g) , resulting in a very high correlation between those titers (Pearson's R=0.9143, p<0.00001).

Low-cost serological assays for COVID-19 are urgently needed in order to enable public health actions, especially in low-income countries. The ELISA assay developed herein meets this requirement: the cost of the assay in terms of consumables was calculated to be in the range of two dimes per sample. However, considering the use in epidemiological surveillance studies, especially in low-income countries, traditional blood sample collection and serum separation would imply the need for clinical laboratory services and refrigerated sample storage and transport that could jeopardize the advantages of the ELISA test. To overcome this critical limitation, we devised a simple storage and transport of blood samples collected by finger prick in filter paper strips to allow sampling in remote regions, or regions lacking a laboratory network. For that, the assay was further standardized to work using eluates from whole blood collected in filter paper (Fig 2h) , a classic protocol that permits titration of antigen immunoreactivity [5] . Plastic strips with filter pads could be even more convenient for sampling and storing for retesting (Fig 2k) . A first comparison of titration curves using serum samples or dried blood spots (DBS) in filter paper displayed similar results (Figs 2i-2j) . O.D. ratios obtained for serum samples, and DBS either in filter paper or in pads showed very high correlation (Figs 2l-2m), as well as high reproducibility among pads from a given strip (Fig 2n) . By using dried blood spots, the low cost of the test is warranted. On top of the two dimes related to consumables costs per test, labor, equipment and logistics costs must be taken into consideration. These vary significantly from country to country, but we estimate that the final cost including all steps from sample collection to test result does not exceed USD 0.50 per test. Compared to USD 100.00 tests available to the customer in the US, the S-UFRJ ELISA is approximately 200-fold less expensive.

Importantly, blood samples collected in filter paper and kept in sealed plastic bags can be preserved for at least 2 months without altering their serological result (data not shown). As such, the S-UFRJ ELISA, comprising the use of eluates from whole blood finger pricks as samples, allows broad serological surveillance in populations regardless of their geographical and socio-economic aspects. As recently shown for the case of Spain [6], population-based sero-epidemiological studies are essential tools to inform authorities if specific public health measures . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 15, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 15, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 15, 2020. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 15, 2020. . https://doi.org/10.1101/2020.07.13.20152884 doi: medRxiv preprint Beckman Coulter), whereas glucose and lactate concentrations were monitored using a metabolite analyzer (YSI 2700, Yellow Springs Instruments). Presence of S protein in the supernatants was determined by spot blots: 3 µL of each sample was applied to nitrocellulose membranes, serum of SARS-CoV-2 convalescent patients (1:500) was used as primary antibody, followed by incubation with anti-human IgG(Fc)-HRP conjugate (Sigma, # SAB3701282) and finally chemiluminescent ECL reagent (BioRad).

Cell suspension harvested from cell cultures was clarified by filtration using 0. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 15, 2020. . https://doi.org/10.1101/2020.07.13.20152884 doi: medRxiv preprint of PBS. At the end, the plate was developed with TMB (3,3',5,5;tetramethylbenzidine) (Thermo Fisher). The reaction was stopped with 50 µL of 1 N HCl, and the optical density (OD) was read at 450 nm with 655 nm background compensation in a microplate reader (BioRad).

According to a protocol approved by the national ethics committee (CONEP, Brazil; protocol #30161620000005257; review #3953368), subjects were initially interviewed and, if they accepted to participate, they signed the informed consent, answered a questionnaire (addressing demographic data, onset and type of symptoms, history of travel abroad, among other information) and had blood (venous blood and/or finger prick) and nasopharyngeal swab collected. Only symptomatic subjects who presented at least two of the following symptoms were included: loss of taste or smell, fever, shortness of breath, diarrhea, headache, extreme tiredness, dry cough, sore throat, runny or stuffy nose, or muscle aches. Dried blood spots (DBS) were obtained by finger pricking with commercially available lancing devices. A 2.5 cm (W) x 7.5 cm (L) filter paper with three blood spots from the same volunteer and commercially available paper hole punching device were used to make dry-blood filter spots, from which blood was eluted for ELISA testing.

In order to determine the titers of neutralizing antibodies in sera samples, serum samples were first heat-inactivated at 56°C for 30 min, and two-fold serial dilutions were incubated with 100 PFU of SARS-COV-2 (GenBank # MT126808. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 15, 2020. . https://doi.org/10.1101/2020.07.13.20152884 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 15, 2020. . . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 15, 2020. . https://doi.org/10.1101/2020.07.13.20152884 doi: medRxiv preprint

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