key: cord-0950114-zei573nr
authors: Narayanaswamy, V.; Pentecost, B.; Alfandari, D.; Chin, E.; Minor, K.; Kastrinakis, A.; Lieberman, T.; Arcaro, K. F.; Leftwich, H.
title: Humoral and cell-mediated response in colostrum after exposure to severe acute respiratory syndrome coronavirus 2
date: 2021-01-04
journal: nan
DOI: 10.1101/2021.01.03.20248715
sha: 5e84318f8d7f03c3824913b6ad36229ba9020fd8
doc_id: 950114
cord_uid: zei573nr

Background: Colostrum provides an immune sharing between a mother and her infant. The transfer in colostrum of antibodies against SARS-CoV-2 and the elicited cytokines may provide crucial protection to the infant. There is limited literature on the immune response to SARS-CoV-2 present in colostrum. Objective: To evaluate the presence of antibodies specific to SARS-CoV-2 and the associated cytokines in colostrum from women who tested positive for the virus. Study Design: Between March and September 2020 we obtained bilateral colostrum samples collected on spot cards within 48 hours of delivery from 15 new mothers who had previously tested positive for SARS-CoV-2. Five of these 15 COVID-19 positive women also provided bilateral liquid colostrum within 1-2 days of providing the spot card samples. Archived bilateral colostrum samples collected from 8 women during 2011-2013 were used as pre-COVID-19 controls. All samples were tested for reactivity to the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike protein using an ELISA that measures SARS-CoV-2 RBD-specific IgA, IgG, and IgM, and for concentrations of 10 inflammatory cytokines (IFN {gamma}, TNF , IL-1 {beta}, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13) using a multiplex electrochemiluminescent sandwich assay. Results: Bilateral colostrum samples from 73%, 73% and 33% of the 15 COVID-19 mothers exhibited IgA, IgG, and IgM reactivity to RBD respectively. Colostrum samples from two of the 8 pre-pandemic controls showed IgA and IgG reactivity to RBD. Additionally, COVID-19 mothers had significantly higher levels of 9 of the 10 inflammatory markers (all except IFN {gamma}) as compared to the pre-COVID-19 controls. Comparable results were obtained with both the spot card-eluates and liquid samples. Conclusions: A strong humoral immune response is present in the colostrum of women who were infected with SARS-CoV-2 before delivering. High levels of 9 inflammatory markers were also present in the colostrum. The evolution and duration of the antibody response, as well as dynamics of the cytokine response, remain to be determined. Our results also indicate that future large-scale studies can be conducted with milk easily collected on paper spot cards.

Study participants were patients at UMass Memorial Medical Center (UMMC, Worcester, MA) 77 and provided consent in accordance with an IRB-approved protocol (H00020140). Fifteen 78 participants who tested positive, and one participant (P01) who tested negative for the CoV-2 RNA, provided bilateral colostrum on the day of, or the day after delivery. Participants 80 hand-expressed colostrum from each breast onto spot cards (Whatman® FTA® card, Millipore 81 Sigma, #WHAWB120205); which were left to dry at room temperature (RT).

Of the 16 participants who provided bilateral colostrum on spot cards, six participants 83 also provided liquid bilateral colostrum within two days after providing the spot card samples. Processing bilateral colostrum samples on spot cards 95 Discs (6 mm diameter) prepared from spot cards were heat-treated for 30 minutes at 56°C to 96 inactivate any virus and were then resuspended in 500 µL of Tris-buffered saline with 0.1% 97 Tween 20 ® (TBST) in a 24-well plate. The plate was incubated with gentle shaking overnight at 98 4°C after which the TBST-eluates were used for detection of anti-SARS-CoV-2-specific 99 . 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 January 4, 2021. ; https://doi.org/10.1101/2021.01.03.20248715 doi: medRxiv preprint 6 #A9941) diluted at 0.2 mg/mL in 0.1 M Sodium Acetate pH 4.5 at 37 0 C for 30 minutes. Known 123 concentrations of anti-Spike-RBD-human (h) IgG1, -hIgM and -hIgA1 (InvivoGen, San Diego, 124 CA, #C3022) were assayed in the ELISAs. The concentration of the highest standard was 1250 125 ng/mL; subsequent standards were prepared by 10-fold serial dilutions starting from 500 ng/mL 126 to 0.05 ng/mL. After background subtraction, concentration curves for IgA, IgG and IgM were 127 generated using a four-parametric logistic (4PL) curve with Excel's Solver Concentrations of unknown samples were calculated using the 4PL equation. Table S1 ). Samples and standards were run in technical duplicates. Welch's t-test was used to assess differences in age and BMI between donations made during 139 2020 and during 2011-2013. Student's t-test was used to compute differences between cytokines. 

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Demographic characteristics of the 24 women are summarized in Table 1 , stratified by the 147 period of colostrum donation: '2020' (COVID-19) versus '2011 -2013 148 controls). Groups did not differ significantly by age or BMI. Colostrum collected during 2011-149 2013 was exclusively from women who identified as White (one woman did not provide 150 information on race). Women who provided colostrum in 2020 were more diverse: they self-151 identified as 31% Hispanic, 13% White Hispanic, 50% non-Hispanic White, and one woman 152 identified as Asian American. 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 January 4, 2021. ; https://doi.org/10.1101/2021.01.03.20248715 doi: medRxiv preprint 8 SARS-CoV-2 (COVID-19 pos ) tests, and one woman (P01) who tested negative for SARS-CoV-2. 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.

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( Table 2 ). In contrast, among the pre-COVID-19 controls, only one analyte, IL-8, was detected 214 in all samples ( Table 2) . 215 Cytokine concentrations were higher among COVID-19 pos participants as compared to 216 pre-COVID-19 controls, and again this was the case for both liquid colostrum and spot card-217 eluates (except for IFNg and IL-6, which were not higher in the spot card). However, the 218 concentration of cytokines was higher in the liquid colostrum than in the spot card-eluates for all 219 analytes ( Table 2 ). Because we did not have a dilution factor associated with the spot card 220 extraction, statistical comparison between COVID-19 pos and pre-COVID-19 samples was limited 221 to the liquid colostrum.

Among liquid samples, significantly elevated concentrations for 9 of 10 cytokines were 223 observed in the COVID-19 pos group (2020) (Figure 3 ). Only IFNg was not significantly higher.

Moreover, there is an indication that cytokine levels in bilateral colostrum obtained from 225 symptomatic participants were higher (red-filled circles; Figure 3 ) compared to levels in 226 asymptomatic participants. This distinction was particularly clear for IL-2, IL-4, IL-6, IL-10 and 227 IL-12. Additionally, we explored the association between antibody response and cytokine levels 228 and found that SARS-CoV-2-specific IgA, IgG and IgM were negatively correlated to IL-13 in 229 spot card-eluates (Table 3) . This pattern was not detected in liquid colostrum from pre-COVID-230 19 controls (Table S2) . 

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The copyright holder for this preprint this version posted January 4, 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.

The copyright holder for this preprint this version posted January 4, 2021. ; 12 of 229E coronavirus 4 . Alternatively, Isho et al also observed elevated saliva IgA and IgG 260 reactivities to SARS-CoV-2-RBD among their pre-COVID-19 controls, but did not attribute the 261 response to a prior coronavirus infection 21 . We were concerned that the reactivity in the left 262 breast of P20 could have been due to experimental error, however, a repeat analysis confirmed 263 the IgA and IgG reactivities. consented to provide spot card followed by liquid colostrum. That the liquid sample was 280 collected from only 6 participants is likely due to the smaller volume requested for the spot card,

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The copyright holder for this preprint this version posted January 4, 2021. ; https://doi.org/10.1101/2021.01.03.20248715 doi: medRxiv preprint 13 and greater ease for the staff, as the spot card can be left to dry at RT for several hours, while the 282 liquid colostrum needs to be quickly frozen. 

Given that the differences between the spot card and liquid samples for IgA, IgG and IgM 296 reactivities to RBD are not all unidirectional, we can assume that differences do not simply 297 reflect collection method. Indeed, the differences between spot card and liquid reactivities for 298 P04 could demonstrate antibody evolution in colostrum: i.e., a switch from high IgA to high IgM 299 (Figure 2A) . 300 Cytokine concentrations are significantly higher in the liquid colostrum from COVID-301 19 pos as compared to pre-COVID-19 participants (Figure 3) suggesting a SARS-CoV-2-specific 302 response. However, there is concern that cytokines could be degraded in archived samples. An 303 . 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 January 4, 2021. Our study is among the first to demonstrate the presence of SARS-CoV-2-specific antibodies in 314 colostrum and describes for the first time elevated cytokines in colostrum from women exposed 315 to SARS-CoV-2. The evolution and duration of the antibody response as well as dynamics of the 316 cytokine response remain to be determined. Given the feasibility of the collection method, and 317 the ability to detect antibodies and cytokines, our results indicate that future large-scale studies 318 can be conducted with milk easily collected on paper spot cards. 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 January 4, 2021. We are grateful to all participants who donated colostrum for this study.

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The copyright holder for this preprint this version posted January 4, 2021. ; https://doi. org/10.1101 org/10. /2021 18 cytokines in colostrum of nursing mothers at the extremes of reproductive age. PLoS One. 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 January 4, 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.

The copyright holder for this preprint this version posted January 4, 2021. ; https://doi.org/10.1101/2021.01.03.20248715 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.

The copyright holder for this preprint this version posted January 4, 2021. ; https://doi.org/10.1101/2021.01.03.20248715 doi: medRxiv preprint Concentration is pg/mL for all analytes. If both technical replicates were above the lower limit of detection (LLOD), the final concentration was the mean of the two levels. If both technical replicates were below the LLOD, the final concentration was computed as LLOD divided by the square root of 2 29 1 = Bilateral colostrum provided by 15 COVID-19-positive participants. 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 January 4, 2021. 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 January 4, 2021. values for all analytes indicate bilateral colostrum provided by participant P05 who did not 459 exhibit COVID-19-related symptoms but reported having influenza 3 months before delivery.

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The copyright holder for this preprint this version posted January 4, 2021. ; https://doi.org/10.1101/2021.01.03.20248715 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 January 4, 2021. ; https://doi.org/10.1101/2021.01.03.20248715 doi: medRxiv preprint 

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