key: cord-261855-qpbgq5d8
authors: Walker, Susanne N.; Chokkalingam, Neethu; Reuschel, Emma L.; Purwar, Mansi; Xu, Ziyang; Gary, Ebony N.; Kim, Kevin Y.; Schultheis, Katherine; Walters, Jewell; Ramos, Stephanie; Smith, Trevor R.F.; Broderick, Kate E.; Tebas, Pablo; Patel, Ami; Weiner, David B.; Kulp, Daniel W.
title: SARS-CoV-2 assays to detect functional antibody responses that block ACE2 recognition in vaccinated animals and infected patients
date: 2020-06-18
journal: bioRxiv
DOI: 10.1101/2020.06.17.158527
sha: 
doc_id: 261855
cord_uid: qpbgq5d8

SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) has caused a global pandemic of COVID-19 resulting in cases of mild to severe respiratory distress and significant mortality. The global outbreak of this novel coronavirus has now infected >8 million people worldwide with >2 million cases in the US (June 17th, 2020). There is an urgent need for vaccines and therapeutics to combat the spread of this coronavirus. Similarly, the development of diagnostic and research tools to determine infection and vaccine efficacy are critically needed. Molecular assays have been developed to determine viral genetic material present in patients. Serological assays have been developed to determine humoral responses to the spike protein or receptor binding domain (RBD). Detection of functional antibodies can be accomplished through neutralization of live SARS-CoV2 virus, but requires significant expertise, an infectible stable cell line, a specialized BioSafety Level 3 (BSL-3) facility. As large numbers of people return from quarantine, it is critical to have rapid diagnostics that can be widely adopted and employed to assess functional antibody levels in the returning workforce. This type of surrogate neutralization diagnostic can also be used to assess humoral immune responses induced in patients from the large number of vaccine and immunotherapy trials currently on-going. Here we describe a rapid serological diagnostic assay for determining antibody receptor blocking and demonstrate the broad utility of the assay by measuring the antibody functionality of sera from small animals and non-human primates immunized with an experimental SARS-CoV-2 vaccine and using sera from infected patients.

options for rapid diagnostic of functional antibody responses, fast and simple functional assays 71 may prove to be a critical assessment tool to discriminate between potential CPT donors.

In parallel with CPT, major academic, industry and government entities are pushing for 

Pseudovirus neutralization assays run in BSL-2 facilities were quickly developed to detect the 91 functional antibody response in sera(21). While this is a critical tool for determining protective 92 antibody titers, it requires several days for a readout and are not standardized between 93 laboratories. The pseudoviruses produced in these assays are not easily manufactured and 94 take time to express, harvest, and titer. One such approach to help augment the methods listed 95 above is an enzyme-linked immunosorbent assay (ELISA) employed in a competitive manner to determine levels of ACE2 receptor blocking antibodies in a sample. In addition, recent advances 97 of portable and field-deployable surface plasmon resonance (SPR) devices(22) and widespread 98 availability of SPR instruments in research laboratories make SPR an additional platform for 99 measuring ACE2 receptor blocking. Here, we describe a competition ELISA assay and a SPR 100 assay developed to rapidly detect ACE2 receptor blocking antibodies in IgGs and sera of 101 vaccinated mice, guinea pigs, rabbits and non-human primates, as well as, human samples (Fig. 1B) . We next sought to confirm the 116 functionality of ACE2-IgHu. Previous studies suggest SARS-CoV-2 binds to ACE2 with an 117 affinity range of 4-34nM(6, 11). We determined that our ACE2-IgHu binds with similar affinity to 118 the receptor binding domain (RBD) of SARS-CoV-2 spike (27.5nM) as assessed by SPR (Fig.   119   1C ). Next, using Enzyme-Linked Immunosorbent Assays (ELISAs) we immobilized full-length 120 SARS-CoV-2 spike protein (containing both the S1 and S2 subunits) and incubated a dilution 121 series of ACE2-IgHu (Fig. 1D) . The binding curves confirmed the high affinity interaction of the receptor for the spike protein. We further showed similar binding for two independent batches of 123 ACE2, as well as a sample that was frozen and thawed (Fig. S1A) . From the binding curve, we 124 hypothesized that a reasonable concentration of ACE2 protein fusion needed to see competitive 125 blocking while still binding >90% of immobilized spike protein in the absence of blocking would 126 be around 0.1-1 ug/ml (Fig. 1D, red arrow) .

To examine if we could construct a competition assay, we employed ACE2-IgMu (mouse Fc) to 128 act as a competitor to ACE2-IgHu. To match our initial binding ELISA format, the competition 129 ELISA assay similarly captured a His6X-tagged full-length spike protein by first immobilizing an 130 anti-His polyclonal antibody. A dilution series of the competitor (ACE2-IgMu) was pre-mixed with 131 a constant concentration of soluble receptor (ACE2-IgHu). A secondary anti-human antibody 132 conjugated to horseradish peroxidase (HRP) determines the amount of ACE2-IgHu present 133 through a TMB colorimetric readout ( Fig. 2A) . In order to formally determine the optimal 134 concentration of soluble receptor (ACE2-IgHu), we performed the assay at four concentrations 135 ranging from 0.01ug/ml to 10 ug/ml (Fig. 2B) . The ACE2-IgHu concentration of 0.1 ug/ml (red 136 curve in Fig. 2B) , was able to show a complete inhibition curve in the presence of ACE2-IgMu.

Animal IgG and serological competition

The proof-of-concept competition ELISA displayed a full blocking curve, so we sought to utilize 140 this assay for animals immunized with SARS-CoV-2 spike protein. The same design for the 141 competition ELISA was used for this assay, replacing the ACE2-IgMu competitor with antibodies 142 induced by vaccination (Fig. 3A) . In our previous work, BALB/c mice were immunized with DNA 143 plasmids encoding SARS-CoV-2 spike protein(23). To examine the activity of antibodies in the 144 sera, IgGs from either naïve mice or vaccinated mice 14 days post-immunization were purified 145 using a protein G column. Unlike the ACE2 control which only binds to the receptor binding site 146 (RBS) on the receptor binding domain (RBD) of the spike protein, antibodies from immunized 147 mice can bind to a multitude of epitopes on the spike protein including epitopes on the S1 subunit(which includes the RBD), S2 subunit or S1-S2 interfaces. While antibodies distal to the 149 RBS should have less effect on ACE2 binding, we hypothesized that such distal antibodies may 150 inhibit ACE2 binding directly by sterically obscuring the RBS or indirectly by causing allosteric 151 conformational shifts in the spike protein. To test this, we immobilized either the full spike 152 protein (S1+S2) or S1 alone to examine the levels of detectable blocking antibodies. A mixture 153 of ACE2-IgHu at a constant concentration of 0.1 ug/ml and a dilution series from a vaccinated 154 mouse IgG (IgGm1) or naïve mice IgG (naïve IgGm) was incubated on the plate. An anti-155 human-HRP conjugated antibody was added to determine the ACE2 binding in the presence of 156 IgGm. As Fig. 3B illustrates, there is greater antibody blocking with the full spike protein than 157 with the S1 subunit alone (Fig. S2A ).

To show the utility of this assay in samples from larger mammals, we examined receptor 160 blocking of rabbit sera from SARS-CoV-2 immunization studies. We pooled sera from five 

For full, uninhibited ACE2 binding, the AUC will be larger than the AUC for a competitive curve 166 (Fig. 3C) . As seen with the mouse IgGs, the pooled vaccinated rabbit IgG displayed statistically 167 significant blocking of ACE2 receptor binding compared to the naïve animal pool and the Day 0 168 pool (Fig. 3D, Fig S2B) . The high dose rabbits reduced ACE2 signal relative to the low dose 169 group, highlighting the utility of the assay to help discriminate between different vaccine 170 regimens. Up to this point we have analyzed purified IgGs collected from sera, however we 171 wanted to validate the use of this assay on serological samples as well. The same rabbit sera 172 pools were used as competitors in the competition ELISA assay in a dilution series to compare blocking between sera and purified IgG. The rabbit sera displayed statistically significant ACE2 174 receptor blocking as we saw in the purified IgG assay (Fig. 3E, Fig. S2C ).

Next, we sought to show that we could assess receptor blocking in a third animal model of 177 guinea pigs which were immunized with a SARS-CoV-2 Spike-based vaccine(23). We 178 compared a pool of sera collected on day 14 post immunization to a Day 0 pool (Fig. 3F) . The 183 spike showed statistically significant ACE2 blocking and, importantly, the pooled sera was 184 comparable to the average AUC from all six sera samples (Fig. 3F, Fig. S2D ). The competition 185 ELISA was used to analyze both the IgGs and the sera from the groups, both groups showed 186 statistically significant blocking of the ACE2 receptor in these assays (Fig. S2E, Fig. S2F ). for detection of ACE2 binding in the presence of primate antibody inhibitors (Fig. 4A) . To 196 confirm the function of the replacement ACE2-IgMu, an initial binding ELISA was performed 197 (Fig. 4B) . We predicted a similar concentration was needed for optimal competition on the 198 binding curve (Fig. 4B , red arrow) and confirmed this by running a competition ELISA assay using ACE2-IgHu as the competitor at varying ACE2-IgMu constant concentrations (Fig. 4C Fig. 4F, Fig S4B, S4C ). This finding is consistent with the ACE2 blocking data and we 211 show a correlation between pseudovirus neutralization ID50 and AUC for residual ACE2 212 blocking across all our datasets (Fig. 5) . Thus, we have demonstrated that the ACE2 213 competition assay can be employed to measure receptor inhibition levels of human samples.

To quantitate blocking of the Spike-ACE2 bimolecular interaction in a second, independent 217 experiment, we developed a sensitive surface plasmon resonance (SPR) assay. SPR is a 218 widely used platform that does not require secondary antibodies and therefore we could use a 219 single assay format for small animals, NHPs and humans. In our assay, a CAP sensor chip is 220 used to capture single stranded DNA coupled to streptavidin. We are then able to capture 221 biotinylated spike protein to the surface of the sensor chip (Fig. 6A) . In SPR, changes in 

To demonstrate the feasibility of this assay, we used ACE2-IgHu as both the sample 228 (ACE2 sample ) and the receptor (ACE2 receptor ). The sensorgram for this experiment shows 229 ACE2 sample injections at various concentrations binding to SARS-CoV-2 RBD between 0 and 125 230 seconds ( Fig. 6B) . At 225 seconds we inject ACE2 receptor at 100nM. We observe ACE2 receptor 231 binding to SARS-CoV-2 RBD at the lower ACE2 sample concentrations, but not at the highest 232 ACE2 sample concentration. The binding signals of ACE2 sample and ACE2 receptor intersect close to 233 100nM, suggesting the assay is working as expected (Fig 6C) . A measure of % ACE2 receptor 234 inhibition can be calculated to the dose dependence response of the sample (Fig. 6D) . While 

The Spike-ACE2 interaction is also being considered as an important therapeutic target. Indeed,

there have been SARS-CoV-1 Spike-ACE2 inhibitors developed previously(29). To examine the 280 functionality of these small molecules beyond direct binding to the spike protein, assays such as 281 the one developed here are needed. The SPR instrument is often used for drug discovery and 282 the SPR assay could be easily adapted to examine blocking capabilities of candidate drugs. The

ELISA assay does not depend on the molecular identity of the competitor, so small molecule or 284 peptide inhibitors could be directly assessed in this assay.

Our study presents a new set of assays for assessing ability of antibody samples to inhibit 287 SARS-CoV-2 Spike interaction with its receptor. As with most assays, the limit of detection can 288 be an issue. In some of our samples, we saw robust blocking and in others there was minimal 289 blocking. This could be a property of the samples themselves or a limit in the ability to detect 290 ACE2 inhibition in our assays. In addition, discovering functional monoclonal antibodies can be 

The SARS-CoV-2 pseudovirus was produced by co-transfection of HEK293T cells with 1:1 ratio 472 of DNA plasmid encoding SARS-CoV-2 S protein (GenScript) and backbone plasmid pNL4- 

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Animal IgG and serological competition. a) AUC is significantly 665 decreased in the presence of vaccinated mouse IgG competitors; however a greater decrease 666 is observed when full-length CoV-2 spike protein was immobilized versus naïve mouse IgG 667 samples. b) ELISA competition curves for vaccinated rabbit IgG (IgGr low dose, blue; IgGr high 668 dose, red) or sera (sera low dose, blue; sera high dose, red) versus naïve rabbit IgG or c) sera 669 samples (grey) and pooled Day 0 rabbit IgG or sera samples (black). d) ELISA competition 670 curves for Week 2 vaccinated guinea pig sera (pool, dark blue; individual animals, blue) versus 671 naïve guinea pig sera samples (grey) and pooled prevaccinated guinea pig sera samples 672 (black). e) AUC for vaccinated guinea pig IgG pool (blue) versus naïve guinea pig

Primate serological competition. a) Four constant concentrations of 677 ACE2-IgMu were tested with varying concentrations of the ACE2-IgHu competitor to establish 678 an optimal ACE2-IgMu concentration which displays a full blocking curve (red, 1.0ug/ml) from 679 the competitor dilution series while retaining a wide range in signal. b) ELISA competition 680 curves for vaccinated NHP sera (blue) versus

Human sera from nine SARS-CoV-2 positive COVID-19 patients 683 was tested in the primate competition assay and compared to sixteen naïve human sera 684 collected pre-pandemic. The AUC of the COVID-19 patient serum (purple) is significantly 685 decreased compared to the pre-pandemic human serum (grey) and normalized to a buffer 686 control. b) pseudovirus neutralization assay for the nine

The authors would like to Matthew Sullivan for providing feedback on the manuscript.

Funding