key: cord-0718110-kcv1bmyx authors: Zou, J.; Bretin, A.; Gewirtz, A. title: Antibodies to SARS/CoV-2 in arbitrarily-selected Atlanta residents date: 2020-05-06 journal: nan DOI: 10.1101/2020.05.01.20087478 sha: 523dcf40ff10ffe2f1d7f0677a582eb25f7730e3 doc_id: 718110 cord_uid: kcv1bmyx We quantitated anti-SARS/CoV-2 IgG and IgM by ELISA in self-collected blood samples (n=142) in arbitrarily-selected metro Atlanta residents, primarily acquaintances of the authors' lab members from 4/17-4/27, 2020. Archived serum (n=34), serum from nucleic acid test (NAT)-positive subjects (n=4), and samples collected from NAT-positive community members (n=4) served to validate the assay. The range of anti-SARS/CoV-2 antibodies in archived and NAT-positive sera indicated need to compromise sensitivity or specificity. Accordingly, we set a cutoff of 4 SD above the mean for IgG and 3 SD above the mean for IgM to indicate that an individual had been exposed, and developed some degree of immunity, to SARS/CoV-2. The IgG cutoff clearly compromised sensitivity but offered high specificity, both of which were harder to gauge for IgM. Based on these cutoffs, excluding subjects whose participation resulted from self-suspected SARS/CoV-2 infection, we found 7.1% positivity for anti-SARS/CoV-2 IgG (3 of 127 subjects) or IgM (6 of 127). While we do not claim this small immune survey is broadly representative of metro Atlanta, and we have greater confidence in the IgG results, which had only 2.4% positivity, it nonetheless demonstrates that persons with antibodies to SARS/CoV-2, who've not suspected they'd been exposed to this virus, can readily be found in various Atlanta area neighborhoods (9 positives were in 8 zip codes). Accordingly, these results support the notion that dissemination of the virus is more widespread than testing would indicate but also suggests that most persons in metro Atlanta remain vulnerable to this virus. More generally, these results support the general utility of sero-surveillance to guide public policy but also highlight the difficulty of discerning if individuals have immunity to SARS/CoV-2. from self-suspected SARS/CoV-2 infection, we found 7.1% positivity for anti-SARS/CoV-2 IgG 48 (3 of 127 subjects) or IgM (6 of 127). While we do not claim this small immune survey is 49 broadly representative of metro Atlanta, and we have greater confidence in the IgG results, 50 which had only 2.4% positivity, it nonetheless demonstrates that persons with antibodies to 51 SARS/CoV-2, who've not suspected they'd been exposed to this virus, can readily be found in 52 various Atlanta area neighborhoods (9 positives were in 8 zip codes). Accordingly, these results 53 support the notion that dissemination of the virus is more widespread than testing would indicate 54 but also suggests that most persons in metro Atlanta remain vulnerable to this virus. More 55 generally, these results support the general utility of sero-surveillance to guide public policy but 56 also highlight the difficulty of discerning if individuals have immunity to SARS/CoV-2. 57 58 . 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) There is now a wide interest in measuring SARS-CoV-2 antibodies both as a means of gauging 60 the true extent to which persons within a particular geographic region have been infected by this 61 virus and perhaps as a means of discerning whether individuals might now be immune from 62 SARS/CoV-2-induced disease. To address the former, some have begun conducting "sero-63 surveys". One recently publicly-posted study described use of a lateral flow test, which purports 64 to give a qualitative yes or no for presence of absence of such antibodies and observed that 4.3 % 65 of tested resident of Santa Clara CA had been exposed to the virus [1] . Press releases of other 66 studies that did not give details of their methodology observed presence of SARS-CoV-2 67 antibodies in 14.3% of resident in Heinsberg, Germany and 18% of residents in New York State. 68 Herein, we report results of our effort to measure SARS/CoV-2 antibodies in resident of 69 metropolitan Atlanta, GA. We analyzed self-collected blood samples from acquaintances of the 70 authors' lab members by a quantitative lab-based ELISA method. We found that antibodies to 71 SARS/CoV-2 are not merely a yes or no but have a broad quantitative range, with some overlap 72 when comparing samples from confirmed infected subjects versus samples collected before the 73 advent of SARS-CoV-2. Nonetheless, our quantitative approaches enabled seemingly readily and 74 reliable discernment that about 3 and 6 of 127 arbitrarily subjected Atlanta area residents 75 displayed SARs/CoV-2-specific IgG and IgM, respectively, which likely reflects that they have 76 been exposed to this virus. 77 78 . 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 May 6, 2020. . https://doi.org/10. 1101 /2020 Board and/or Biosafety committee. 82 83 Subject selection: An advertisement was prepared that explained that the goal of the study was 84 to perform community by sero-surveillance, and specified that no results would be provided to 85 individual participants. The advertisement was circulated by the authors and their lab members in 86 their neighborhoods (see table 1 for subject zip codes), which resulted in enrollment of 142 87 individuals, 135 of whom had not been tested for SARS/CoV-2 and 2 individuals whom had 88 suspected they'd been infected but tested negative at an area hospital. While these subjects were 89 not asked any health-related questions, some volunteered that they suspected they might have 90 been previously infected, which we noted. Additionally, we enrolled 4 community members 91 whom had been referred to the study by word of mouth primarily because they had been deemed 92 positive for SARS/CoV-2 by a PCR-based test. One of these subjects recruited 5 household 93 members, one of whom exhibited symptoms. We also enrolled 4 subjects who had not been 94 tested for SARS/CoV-2 but yet were referred to the study because they had told associates of the 95 study team that they believed they had been infected by SARS/CoV-2. Additionally, 3 serum 96 samples from NAT-positive subjects were provided by Sean Stowell (Emory University) and one 97 was provided by Mount Sinai School of Medicine Department of Pathology. 98 99 Blood collection and serum isolation: Subjects were provided an autoclaved 1.5 ml Eppendorf 100 tube, 2 26-gauge safety lancets, and asked to watch a video demonstrating collection of 1-2 101 . 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 May 6, 2020. . https://doi.org/10.1101/2020.05.01.20087478 doi: medRxiv preprint blood droplets https://www.youtube.com/watch?v=H9e-9xvhrms. Subjects were advised to then 102 collect their blood and contact study team. This procedure generally resulted in blood being centrifuged (5000 rpm, 10 minutes) and serum isolated in a biosafety cabinet at GSU within 24 104 hours of blood collection, although test comparisons of allowing storing blood for up to 3 days 105 did not seem to impact results. This procedure yielded over 5 and 10 microliters of serum, 106 respectively in over 97% and 93% of subjects respectively. Serum was then heated, in sealed 107 Eppendorf tubes, to 55 o C for 15 minutes to reduces it infective potential. Archived control sera 108 consisted of samples collected from healthy control subjects in the Atlanta area from 2015-2019 109 that had been stored at -80 o C. Such sera was heat-inactivated as above prior to use in this study. 110 Recombinant his-tagged S1 SARS/CoV-2 S1 Spike protein, purchased from Sino Biologicals 113 (Wayne PA), was diluted to a concentration of 1 g/ml in PBS and applied overnight, 100 l per 114 well, to Ni-coated 96-well plates (Fisher/Thermo) at 4 °C . Plates were then blocked with PBS/ 115 5% skim milk for 1h at room temperature, washed 3X with PBST (PBS 0.1% tween 20). Serum, 116 sequentially diluted 1:100 in PBS/1% BSA and 1: 10 in 3% skim milk respectively, was then 117 applied to wells for 1h at 37 °C. Plates were then washed 3X with PBST and then incubated with 118 antihuman IgG or IgM (KPL), which was diluted 1:5000 and 1:50,000 respectively. 45 min 119 later, plates were washed 3X with PBST and developed with TMB for 10 minutes at which time 120 reaction was stopped with stop reagent (KPL, TMB Stop solution) and OD450 nm measured by 121 a 96-well plate reader (subtracting readings at 540 nm). A positive control for the assay was used 122 to assess and correct for plate to plate variance of the assay. In the course, of setting and 123 validating this assay, many samples were assayed multiple times and gave similar relative 124 . 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 May 6, 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 May 6, 2020. . https://doi.org/10. 1101 /2020 thought to have existed, displayed modest but measurable immune reactivity to recombinant 132 SARS/CoV-2 S1 spike protein (Figure 1) , likely reflecting non-specific binding or cross-133 reactivity to other Coronavirus spike proteins. In any case, we presumed that any newly collected 134 serum samples displaying immune reactivity to SARS/CoV-2 spike protein above the range of 135 the archived samples likely reflects that an individual has been exposed to SARS/CoV-2. 136 Conversely, test samples having values within the range of the archived samples reflects an 137 individual's lack of exposure to SARS/CoV-2 or that the antibody response an individual 138 generated at the time of blood collection was not sufficient to rise above the range of the 139 archived samples. Indeed, one study reported that 30% of hospitalized subjects don't make 140 antibodies to the virus [3] . For example, one of the 4 hospital-provided NAT+ serum samples 141 yielded an OD within the range of the control samples. Hence, we concluded that setting a 142 specific cut-off to declare samples SARS/CoV-2 positive or negative will require compromising 143 sensitivity or specificity. Accordingly, as all archived sample fell within 2.6 SD of the group 144 mean, we set a cut-off of 4 SD above that mean as a threshold to indicate that an individual had 145 likely been exposed to SARS/CoV-2. If values had a normal distribution, such a cut-off would 146 provide over 99% specificity. This cut-off result was met by 3 of 4 community-collected NAT+ 147 samples. The IgM assay was harder to validate in that, as one might expect, the 4 hospital-148 provided convalescent serum samples were all within the range of the archived samples. Hence, 149 based on overall analysis of our data and other studies, we set a cutoff of 3 SD to indicate likely 150 recent exposure to SARS/CoV-2. Based on this cutoff, 2 of our 4 community-acquired NAT+ 151 samples, including the one that was negative for IgG, were positive for SARS/CoV-2 anti-IgM. 152 . 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 May 6, 2020. . https://doi.org/10.1101/2020.05.01.20087478 doi: medRxiv preprint Based on the above-defined cutoffs, 7 of all 142 community-collected samples we 155 collected in this study were positive for SARS/CoV-2 IgG. Yet, the level of SARS/CoV-2 IgG 156 in all of these samples was dramatically lower than the 2 high-titer hospital-provided positive 157 control samples. We speculate that severe SARS/CoV-2 infections may result in viremia that 158 drives very high antibody responses whereas cases not requiring hospitalization result in more 159 subtle and/or delayed antibody responses. Accordingly, none of the blood samples we tested 160 (n=20), including 2 IgG+ and 2 IgM+ subjects, showed detectable virus in blood by PCR. Of the 161 7 IgG+ samples we collected from the community, 3 were from NAT+ subjects and one was a 162 household member of one of those subject. This latter sample yielded more than double the OD 163 of the sample provided by the overtly ill NAT+ subject despite the household member lacking 164 severe symptoms. Several other subjects were included in the study purely due to having 165 contacted the study team after having heard of the study second hand. Excluding both groups, 166 results in 3 of 127 subjects that were selected based only on acquaintance to the study team as 167 being IgG+. These positives were from 3 different zip codes (Table 1) 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 May 6, 2020. . https://doi.org/10.1101/2020.05.01.20087478 doi: medRxiv preprint community has acquired some degree of immunity to it. While testing for SARS/CoV-2, which 179 has largely focused on those with symptoms and, as of April 27, 2020, confirmed the presence of 180 the virus in about 0.23% of the population, true incidence of exposure is presumed to be much 181 higher. In accord with this notion, we found that 7.1% of those we arbitrarily-selected, we submit 182 unbiasedly, displayed levels of anti-SARS/CoV-2 antibodies that indicated likely exposure to the 183 virus. It is difficult to determine the extent to which our survey is representative of metro 184 Atlanta in general or even those areas that are most represented. We certainly would not claim 185 that our selection of participants was random but yet we submit our arbitrary selection process 186 had minimal bias in terms of whether persons suspected they'd been infected and largely resulted 187 in inclusion of households that would not have likely had close contact in 2020. But on the other 188 hand, our survey participants were heavily weighted toward northeast Atlanta neighborhoods and 189 surely under-represents African Americans and Latinos, whom are thought to be most likely to 190 be exposed to the virus. Nonetheless, that our small survey identified SARS/CoV-2 antibody-191 positive in subjects whom were Caucasian, African Americans, Latinos, and Asian Americans 192 suggesting the virus has broadly disseminated in the region. The modest numbers of African 193 Americans and Latinos included make it difficult to weight or extrapolate our results but yet 194 attempting to so would lead us to guess that applying our methodology broadly and randomly 195 across the region would yield higher rates of SARS/CoV-2 antibody positivity than observed in 196 our study. 197 198 . 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 May 6, 2020 . . https://doi.org/10.1101 /2020 observed in Santa Clara SC in early April and is considerably lower than that recently measured 200 in NY. Yet, aside from our study being smaller and not having attempted random sampling, our 201 quantitative methodology may have allowed us to detect weak but nonetheless real positives that 202 would seem to be extremely difficult to detect by lateral flow without also picking up many false 203 positives. Considering this presumed methodological differenced (to our knowledge methods 204 used by NY State have not been reported in detail) and that the per capita death rate in New York 205 State is over 10 times that of Georgia, we speculate that applying our methodology to NY would 206 reveal higher rates of exposure than they observed by their methodology. Yet, one can readily 207 imagine alternative explanations. For example, perhaps sequence variants of SARS/CoV-2 208 predominating Atlanta are less virulent than that those in NY. Perhaps the lower population 209 density and relatively low use of public transportation in Atlanta resulted in exposures to lower 210 doses of the virus. 211 During the course of our study, about 15 participants expressed the view that they 212 strongly suspected they had been exposed to SARS/CoV-2 based on symptom; about 10 of our 213 unbiasedly selected subjects and 5 others who contacted us based on their symptoms. Only one 214 of these subjects displayed SARS/CoV-2 antibodies (IgM) thus indicating the difficulty of 215 diagnosing by symptoms. Conversely, none of the 3 IgG+ positive subjects we unbiasedly 216 identified displayed any symptoms, cautioning against presuming healthy people are not 217 infected. Yet, that all 3 had household members lack SARS/CoV-2 antibodies fits with the notion 218 that such individual are not "super spreaders" of the virus. In conclusion, we readily 219 acknowledge our study faced a variety of limitations, as one might expect would be the case 220 when a small team of mouse-based researchers seek to originate a clinical study in the course of 221 . 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 May 6, 2020. . https://doi.org/10. 1101 /2020 3 0 0 30303 1 0 0 30305 3 1 1 30307 15 0 1 30308 6 1 0 30309 1 0 1 30312 3 0 0 30316 1 0 0 30317 6 1 1 30318 1 0 0 30319 3 0 0 30324 3 0 0 30327 4 0 0 30328 2 0 0 30329 4 0 0 30339 1 0 0 30340 1 0 0 30342 2 0 0 30344 1 0 0 30345 1 0 0 30606 2 0 0 31302 1 0 0 241 . 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 May 6, 2020. . https://doi.org/10. 1101 /2020 Nonetheless, we believe our results can inform approaches to sero-surveil 222 COVID-19 Antibody Seroprevalence in SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol for a 229 Serological Assay, Antigen Production, and Test Setup Neutralizing antibody responses to SARS-CoV-2 in a COVID-19 232 reCoVered 233 2 patient cohort and their implications. MedRxix, 2020