*4.2. Enzyme-Linked Immunosorbent Assay (ELISA)*

MaxiSorpTM 96-well plates (Thermofisher) were coated with SARS-CoV-2 RBD-His or SARS-CoV-2 N-His protein (see Supplementary Material for details on clone construction and protein purification) at 50 ng per well diluted in PBS and incubated at 4 ◦C overnight. Following removal of coating solution, plates were blocked with blocking buffer (5% nonfat milk in PBS with 0.1% Tween-20, PBST) and incubated at 37 ◦C for 1 h. Three-fold serial dilutions of samples were prepared in blocking buffer, then incubated on plates at 37 ◦C for 2 h. Plates were washed three times with PBST followed by incubation with donkey anti-human IgG horseradish peroxidase (HRP) conjugated secondary antibody (Jackson ImmunoResearch) diluted 1:10,000 in blocking buffer at 37 ◦C for 1 h. Plates were washed again prior to the addition of TMB peroxidase substrate mix (Seracare) and incubated at room temperature (RT) for 5 min. TMB stop solution (Seracare) was added and the optical density (OD) at 450 nm was measured using a Molecular Devices SpectraMax 340PC Microplate Reader. A normal human serum non-reactive to SARS-CoV-2 was included as

negative control and a human serum known to be reactive to SARS-CoV-2 was included as positive control on each plate.

#### *4.3. Focus Reduction Neutralization Test (FRNT)*

Neutralization assays were performed as previously described [28] except with <sup>2</sup> × <sup>10</sup><sup>3</sup> FFU/mL of SARS-CoV-2 (University of Pittsburgh clinical isolate from June 2020). Fixation was performed using 4% paraformaldehyde for 20 min at RT. Rabbit anti-SARS-CoV-2 N antibody (GenScript custom, 1:3000 diluted in blocking buffer) was used as primary antibody and goat anti-rabbit IgG HRP (Jackson ImmunoResearch, 1:1000 diluted in blocking buffer) was used as secondary antibody. MossBio TMB substrate was used for foci development. The FRNT50 is the dilution of sera that neutralized at least 50% of the input virus.

#### *4.4. Data Analysis*

The US Census 2019 ACS 5-year estimates of the population in Allegheny County, PA, was accessed online and data regarding race, age, sex and median household income based upon zip code was abstracted. All graphs were made using GraphPad Prism Version 9. Correlation coefficient (r) and two-tailed probability (p) values were calculated using Spearman's correlation. Confidence intervals were calculated using the Clopper–Pearson exact method. The endpoint titer was defined as the dilution of the serum that gave an OD value at least three standard deviations above the average value obtained from the negative control serum.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/pathogens10060710/s1, Figure S1: Comparison of RBD and N titers between the in-house assays and that reported by NIBSC using WHO international standards. Five dots shown on each graph represent samples of one healthy donor collected before 2019 and four COVID-19 recovered patients. Spearman's rank correlation coefficient r and probability (*p*) value (two-tailed) are shown. Figure S2: Comparison of income and race between infected and vaccinated cases in February 2021. Median income distribution in infected and vaccinated groups (a): Y axis is the median household income of the area based on the zip code of the home address reported by each study subject. The line in each group represents the median. \* represents a *p* value < 0.05 by an unpaired *t*-test (two-tailed). Race distribution in infected and vaccinated groups (b): Total samples refer to all samples collected in February 2021. Data were compared to that of American Community Survey (ACS) 5-year estimates for Allegheny County, PA, generated by United States Census Bureau. Table S1: Detailed groups of samples used in the Specificity Assays of SARS-CoV-2 RBD and N. Table S2: Demographics of study subjects.

**Author Contributions:** Conceptualization, A.K.M.; validation, L.X.; formal analysis, L.X.; J.D.; investigation, L.X.; J.D.; resources, A.K.M., V.L.S.; W.P.D.; M.R.S.; S.E.W.; writing—original draft preparation, L.X.; writing—review and editing, A.K.M.; visualization, L.X.; supervision, A.W.; W.P.D.; A.K.M.; project administration, D.J.B.; funding acquisition, A.K.M. All authors have read and agree to the published version of the manuscript.

**Funding:** This research was funded by the DSF Charitable Foundation, the University of Pittsburgh Clinical and Translational Science Institute (CTSI), the Burroughs Wellcome (CAMS 1013362.02) and Stanley and Susan Plotkin/Sanofi Pasteur Fellowship Award.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of The University of Pittsburgh.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in study 20030228. Patient consent was waived for study 20040220 since only residual, deidentified samples were used.

**Data Availability Statement:** Data is contained within the article or supplementary material.

**Conflicts of Interest:** The authors declare no conflict of interest.
