key: cord-0758439-ka2kyqmk
authors: Mades, A.; Chellamuthu, P.; Lopez, L.; Kojima, N.; MacMullan, M. A.; Denny, N.; Angel, A. N.; Casian, J.; Brobeck, M.; Nirema, N.; Klausner, J. D.; Turner, F. E.; Slepnev, V. I.; Ibrayeva, A.
title: Detection of persistent SARS-CoV-2 IgG antibodies in oral mucosal fluid and upper respiratory tract specimens following COVID-19 mRNA vaccination
date: 2021-05-07
journal: nan
DOI: 10.1101/2021.05.06.21256403
sha: 4908140f44644b673d10d0d7ce838d4bf5c307d8
doc_id: 758439
cord_uid: ka2kyqmk

Previous studies have shown that mRNA COVID-19 vaccines are highly effective at preventing SAR-CoV-2 infection by generating an immune response, which in part produces SARS-CoV-2 IgG antibodies in serum. In this study, we hypothesized that COVID-19 vaccines may elicit production of SARS-CoV-2 IgG antibodies in the upper respiratory tract, such as in oral and nasal mucosal fluid. To test that hypothesis, we enrolled 114 participants within 3-7 days of receiving the first dose of the Moderna mRNA COVID-19 vaccine and collected oral mucosal fluid samples on days 5, 10, 15, and 20 after each vaccine dose. Of participants naive to SARS-CoV-2 (n = 89), 79 (85.4%) tested positive for SARS-CoV-2 IgG antibodies by time point 2 (10 days +/-2 days after first vaccine dose), and 100% tested positive for SARS-CoV-2 IgG by time point 3 (15 days +/- 2 days after first vaccine dose). Additionally, we collected paired oral mucosal fluid and anterior nares samples from 10 participants who had received both vaccine doses. We found that participants had an average SARS-CoV-2 IgG antibody concentration of 2496.0 +/- 2698.0ng/mL in nasal mucosal fluid versus 153.4 +/- 141.0ng/mL in oral mucosal fluid. Here, we demonstrate detection and longitudinal persistence of SARS-CoV-2 IgG antibodies in upper respiratory tract specimens following COVID-19 mRNA vaccination. A high concentration of IgG targeting viral spike protein in the upper respiratory system may play an unexplored role in the prevention of SARS-CoV-2 infection and deserves further investigation.

preventing COVID-19 disease (1). Serum antibodies that were elicited by mRNA-1273 persisted 50 through 6 months following the second vaccination dose, suggesting lasting protection against 51 COVID-19 (2). We hypothesize that mRNA vaccines may elicit a strong antibody response in 52 the upper respiratory tract at the sites where primary infection occurs and propagates, thereby 53 preventing SARS-CoV-2 infection and transmission. An earlier work from our group 54 demonstrated that SARS-CoV-2 IgG antibodies targeting spike protein S1 and S2 can be 55 reliably detected from self-collected oral fluid among participants previously infected with SARS-56

CoV-2 (3). In the present study, we aimed to 1) assess whether SARS-CoV-2 IgG antibodies 57 targeting the spike protein are detectable in self-collected oral and/or nasal mucosal following 58 COVID-19 mRNA vaccination; and 2) quantify SARS-CoV-2 IgG antibodies in oral mucosal fluid CC-BY-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 7, 2021. Collection Device (OSCD) for SARS-CoV-2 IgG antibody detection, and a second sample was 91 collected using the Curative oral fluid swab test for SARS-CoV-2 RNA detection. Upon 92 enrollment, participants were trained to self-collect specimens using each collection device 93 while observed by study staff. Participants were provided additional test kits and asked to self-94 collect one of each specimen type unobserved at each follow-up time point, with a two-day 95 grace period for sample collection and two day grace period for returning specimens to the 96 study team. 97 98 63 days +/-5 days after the second COVID-19 vaccination dose, a sub-group of the cohort self-99 collected a paired oral and anterior mades specimen, using the OSCD and nasal swab, 100 respectively, to assess the presence of SARS-CoV-2 IgG antibodies in the anterior nares 101 mucosa following COVID-19 vaccination. Each collection device was weighed prior to and 102 following specimen collection to measure the volume of sample collected. The dilution factor of 103 each specimen was calculated by dividing the volume of preservative fluid (800 µL) by the 104 volume of the sample. The final concentration of SARS-CoV-2 IgG antibodies was determined 105 by multiplying the specimen's determined antibody concentration by the calculated dilution 106 factor. Antibody concentration for other specimens was not adjusted for the dilution factor. 107 108 Between April 2020 and December 2020, we collected oral fluid specimens using the OSCD 109 from participants who were previously infected with SARS-CoV-2 in a separate study over time 110 spanning a six month period. Here, we determined titers of SARS-CoV-2 IgG antibodies in 111 these previously collected oral fluid specimens. 112 . CC-BY-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. A quantitative SARS-CoV-2 IgG ELISA was performed on self-collected oral mucosa specimens 130 collected using the OSCD and anterior nares specimens using the flocked swab. All reagents 131 and proteins were obtained from OraSure® Technologies. 25 μ L of sample diluent and 100 µL 132 of either oral or nasal mucosa specimen were added to 96-well plates coated with both S1 and 133 S2 subunits of the SARS-CoV-2 viral spike glycoprotein. Plates were incubated at ambient 134 temperature for 1h. Sample wells were then washed six times with wash buffer (20x dilution with 135 . CC-BY-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 7, 2021. ; https://doi.org/10.1101/2021.05.06.21256403 doi: medRxiv preprint ddH2O, 350 µL per well) and conjugate solution was added (100 µL per well). Plates were 136 incubated at ambient temperature for 1 h and sample wells were then washed an additional six 137 times. Next, substrate solution was added (100 µL per well) and the plate was incubated at 138 ambient temperature for 30 min. Finally, stop solution was added (100 µL per well). The 139 absorbance of sample wells was measured immediately at 450 nm and 630 nm. Output 140 reports generated the absorbance at 630 nm subtracted from the absorbance at 450 nm. 141

To quantify SARS-CoV-2 IgG antibodies in oral mucosal fluid and nasal samples, an S1-specific 143 monoclonal IgG antibody with no known cross-reactivity to the S2 domain of the spike protein 144 was used as a reference antibody. Cross-reactivity from IgA and IgM were minimal as reported 145 by the manufacturer but was also tested. We confirmed the lack of cross-reactivity from IgA with 146 the absence of detectable IgA signal on an assay. A standard curve was developed using a 147 monoclonal IgG antibody targeting the S1 antigen of SARS-CoV-2 at concentrations of 0, 1.5, 3, 148 6, 12, and 20ng/mL with a polynomial regression curve-fitting model. The standard curve was 149 used to calculate the sample IgG antibody concentration from absorbance values at 450/630 nm 150 from the ELISA assay. The absorbance signal from each sample is directly proportional to the 151 IgG antibody concentration present in the oral fluid (Fig. 1) . Specimens with antibody titer levels 152 exceeding the range of the standard curve were diluted in a sample dilution buffer and re-ran. 153

The Limit of Detection (LoD) was 1 ng/mL and the Limit of Quantification (LoQ) was 1.5 ng/mL. 154

The calculated values for oral fluid specimens presented in this paper represent the diluted 155 specimen, with approximately 800 µL of oral fluid diluted in 800 µL preservative. The real oral 156 fluid IgG antibody concentration is up to several folds larger than the value reported, depending 157 on the volume of fluid collected. 158 . CC-BY-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. identified as female, and 6 (6.3%) had tested positive for SARS-CoV-2 RNA at some time prior 181 . CC-BY-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. . CC-BY-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 7, 2021. ; https://doi.org/10.1101/2021.05.06.21256403 doi: medRxiv preprint Self-collected oral fluid samples using the OSCD from the cohort of unvaccinated participants 227 with a previous SARS-CoV-2 infection (n = 31) were evaluated over time (Fig. 3) sample dilution, we expect the real antibody concentration to be 1.5-2-fold higher than these 246 calculated values. Significantly higher SARS-CoV-2 IgG antibody concentrations were observed 247 in anterior nares specimens compared to OSCD oral fluid specimens, after adjusting for dilution 248 . CC-BY-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. CC-BY-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 7, 2021. ; https://doi.org/10.1101/2021.05.06.21256403 doi: medRxiv preprint COVID-19 vaccines, among older adults, and over a longer study period, to monitor the scale 294 and the duration of the protection against COVID-19 infection via IgG antibodies. 295 296 Testing of the respiratory specimens for SARS-CoV-2 IgG antibody offers a self-collected, non-297 invasive specimen collection alternative to serum specimens that can be scaled with a higher 298 expected patient compliance. 

Efficacy and Safety 325 of the mRNA-1273 SARS-CoV-2 Vaccine

Antibody Persistence through 6 Months after the Second Dose of mRNA-1273 Vaccine 328 for Covid-19

SARS-CoV-2 antibodies in oral fluid obtained using a rapid collection device

Durability of Response after SARS-CoV-2 mRNA-1273 Vaccination

vaccine BNT162b1 elicits human antibody and TH1 T cell responses

Safety and Immunogenicity of SARS-CoV-2 mRNA-1273 Vaccine in Older Adults. N Engl 339

An 341 mRNA Vaccine against SARS-CoV-2 -Preliminary Report

CC-BY-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 7, 2021. CC-BY-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 7, 2021. ; https://doi.org/10.1101/2021.05.06.21256403 doi: medRxiv preprint It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-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) . CC-BY-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 7, 2021. ; https://doi.org/10.1101/2021.05.06.21256403 doi: medRxiv preprint . CC-BY-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 7, 2021. ; https://doi.org/10.1101/2021.05.06.21256403 doi: medRxiv preprint