key: cord-0313195-4eoyruyq authors: Atyeo, C. G.; Shook, L. L.; Brigida, S.; De Guzman, R. M.; Demidkin, S.; Muir, C.; Akinwunmi, B.; Medina Baez, A.; McSweeney, E.; Burns, M.; Nayak, R.; Kumar, M. K.; Patel, C. D.; Fialkowski, A.; Cvrk, D.; Goldfarb, I. T.; Yonker, L. M.; Fasano, A.; Elovitz, M. A.; Gray, K. J.; Alter, G.; Edlow, A. G. title: Maternal immune response and placental antibody transfer after COVID-19 vaccination across trimester and platforms date: 2021-11-14 journal: nan DOI: 10.1101/2021.11.12.21266273 sha: 44e348b6d75c2b043645460ef2ae7e5a749fc8a2 doc_id: 313195 cord_uid: 4eoyruyq The availability of three COVID-19 vaccines in the United States provides an unprecedented opportunity to examine how vaccine platforms and timing of vaccination in pregnancy impact maternal and neonatal immunity. Here, we characterized the antibody profile after Ad26.COV2.S, mRNA-1273 or BNT162b2 vaccination in 158 pregnant individuals, and evaluated transplacental antibody transfer by profiling maternal and umbilical cord blood in 175 maternal-neonatal dyads. These analyses revealed lower vaccine-induced functions and Fc-receptor binding after Ad26.COV2.S compared to mRNA vaccination, and subtle advantages in titer and function with mRNA-1273 versus BN162b2. mRNA vaccinees had higher titers and functions against SARS-CoV-2 variants of concern. First and third trimester vaccination resulted in enhanced maternal immune responses relative to second trimester. Higher cord:maternal transfer ratios following first and second trimester vaccination reflect placental compensation for waning maternal titers. These results support vaccination early in pregnancy to maximize maternal protection throughout gestation, without compromising neonatal antibody protection. Pregnant individuals with COVID-19 are not only at increased risk for severe morbidity and mortality, 1-4 but also for adverse pregnancy outcomes including preterm delivery, pregnancy loss and stillbirth. [5] [6] [7] While vaccination against COVID-19 is a critically important public health strategy to protect pregnant individuals and their pregnancies, only 33% of pregnant individuals have been vaccinated to date. 8 Because pregnant individuals were excluded from initial vaccine clinical trials, [9] [10] [11] data to guide clinical decision-making in this population have lagged behind those for the general population, contributing to vaccine hesitancy. One critical way to increase vaccine confidence in pregnant individuals is through observational data collected from individuals who have received the COVID-19 vaccine during pregnancy. To date, studies have demonstrated that pregnant people mount robust immunological responses to COVID-19 mRNA vaccines (BNT162b2 and mRNA-1273) with final titers achieved being comparable to those in non-pregnant women of reproductive age, [12] [13] [14] and with similar safety and reactogenicity profiles. 12, 14, 15 Several studies of pregnant people receiving COVID-19 mRNA vaccines primarily in the third trimester have also demonstrated the presence of anti-SARS-CoV-2specific antibodies capable of neutralization and immune effector functions in umbilical cord blood at delivery, [12] [13] [14] [16] [17] [18] [19] Maternal vaccination against COVID-19 has the potential not only to protect the pregnant woman, but to confer fetal and neonatal benefit by preventing adverse pregnancy outcomes related to severe maternal COVID-19 illness, and by providing newborns with immunity through transplacental and breastmilk transfer of maternal antibodies. 20, 21 maternal age, gravidity, parity, pre-pregnancy BMI, race, insurance status, obesity, or presence of an autoimmune disorder. Individuals who received the Ad26.COV2.S vaccine were more likely to be of Hispanic ethnicity. We initially evaluated transplacental antibody transfer via systems serology for those participants who had delivered at the time of maternal antibody profiling (n=123 maternalneonatal dyads, Supplemental Table 1 ). There were no differences in gestational age at delivery, mode of delivery, neonatal sex, or neonatal birthweight by vaccine platform. To enhance understanding of transplacental antibody transfer by trimester of vaccination, IgG titers against Spike were quantified using ELISA in these 123 dyads and an additional 52 dyads who had delivered by study completion. In this set of 175 dyads, 27 participants (15%) were vaccinated with Ad26.COV2.S, 62 (35%) with mRNA-1273, and 86 (49%) with BNT162b2. Supplemental Table 2 depicts vaccine type and days elapsed from second dose (or single dose if receiving Ad26.COV2.S) to delivery by trimester of vaccination for this expanded dyad cohort. To begin to understand differences in the vaccine-induced immune response across the three vaccine platforms, we plotted the Spike-specific antibody titer and Fc-receptor (FcR) binding in maternal serum ( Figure 1A and Figure S1A ). These plots reveal that whereas similar antibody profiles against Spike were observed for the two mRNA vaccines (mRNA-1273 and BNT162b2), vaccine-induced antibody titers and FcR-binding across all IgG subclasses and two antibody isotypes (IgG and IgA) were significantly lower in individuals who received the Ad26.COV2.S vaccine. Overall, the anti-Spike response was similar in individuals who received the mRNA vaccines mRNA-1273 and BNT162b2, with the exception of a significantly higher IgG2 anti-Spike response in women vaccinated with mRNA-1273 compared to those vaccinated with All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint BNT162b2 ( Figure S1A ). Individuals who received Ad26.COV2.S also displayed significantly lower antibody functions ( Figure 1B and S1C), as measured by antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), antibodydependent complement deposition (ADCD) and antibody-dependent NK-cell activation (ADNKA, measured as % CD107a+, % MIP-1β+ and % IFNγ+ cells). Antibody titer and FcR-binding against the Spikes from variants of concern (Alpha, Beta, Delta and Gamma) were highly correlated with response to the ancestral Spike, suggesting that individuals who mount a robust vaccine-induced antibody response will have antibodies against variants of concern (Figure S1B). To understand whether the different vaccine platforms elicited antibodies directed at different epitopes of Spike, we plotted the S1-and S2-specific IgG1 and FcR-binding in maternal plasma by vaccine. Interestingly, the IgG1 titer directed against S1 was comparable across vaccine platforms, whereas women who received Ad26.COV2.S had a nonsignificant decrease in IgG1 titer against S2 ( Figure 1C ). FcR-binding against the S1 domain was similar among vaccine platforms, whereas the FcR-binding against S2 was significantly lower for individuals who received Ad26.COV2.S ( Figure 1C ). These data suggest that differences in the FcR-binding of antibodies against S2 are primary drivers of reduced Ad26.COV2.S functions against Spike. Moreover, these data highlight that a single dose of Ad26.COV2.S can induce a similar S1directed response as two doses of mRNA-1273 and BNT162b2. To further examine differences in maternal vaccine response across vaccine platforms, a partial least squares discriminant analysis (PLSDA) was performed. Least absolute shrinkage and selection operator (LASSO) was used to select features most important to the model to prevent overfitting. This analysis revealed that although the vaccine responses in individuals who received mRNA-1273 or BNT162b2 were indistinguishable, the vaccine response in individuals All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint who received Ad26.COV2.S was clearly separated from those who received either mRNA vaccine ( Figure 1D ). Nearly all LASSO-selected features were enriched in the women who received mRNA vaccination ( Figure 1E ). We next sought to determine how the trimester of vaccination impacts the maternal vaccineinduced antibody response. Univariate analyses examining responses by trimester did not reveal trimester-specific differences in anti-Spike antibodies or FcR-binding ( Figure 2A -B, Figure S2A -B). To further investigate the relative contribution of trimester of vaccination to anti-Spike antibody titer, FcR-binding, and function, the mean percentile rank of each feature was plotted by trimester of vaccination ( Figure 2C ). This analysis revealed that both first and third trimester vaccination drove a higher functional antibody response compared to second trimester vaccination, marked by both higher FcR-binding and more functional antibodies as indicated by enhanced ADCD, ADNP, ADCP, and ADNKA responses. Given the observed differences in immune response driven by vaccine platform, we next sought to define the combination of features that best separate vaccine responses by trimester of vaccination within each vaccine platform group. To this end, LASSO was used to pick a minimal set of features that differentiated individuals vaccinated in the second and third trimesters, followed by PLSDA to visualize the separation between the second and third trimesters ( Figure 2D ); due to the smaller sample size, first trimester responses were not included in these analyses. Whereas there was little separation between second and third trimester vaccine responses in women that received the Ad26.COV2.S vaccine (5-fold CV: 0.3), there was a clear separation between the trimesters in women that received the mRNA-1273 vaccines (5-fold CV: 0.89, p < 0.05) and a modest separation between the trimesters in women All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint that received the BNT162b2 vaccine (5-fold CV: 0.73, p < 0.05). The LASSO-selected features show an enrichment of antibody measurements in the third trimester relative to second within mRNA vaccine groups ( Figure S2C-D) . Specifically, individuals who received mRNA-1273 during the third trimester had an enrichment in IgA and IgG2 against variants of concern Alpha and Beta, and enrichment of ADCP compared to those who received mRNA-1273 in the second trimester ( Figure S2C ). This elevation in the IgA and IgG2 response in mRNA-1273 recipients was linked to a highly correlated response across SARS-CoV-2 variants ( Figure S2C ), and the increase in ADCP was strongly correlated with FcR-binding across variants of concern and ADNP activity in these women (S2C). Women who received BNT162b2 in the third trimester had enriched FcR2b-binding and IgM against the Alpha variant, and enriched IgG3 and ADNKA (measured by CD107a expression) responses compared to women who received BNT162b2 in the second trimester ( Figure S2D ). The increase in Alpha FcR2b-binding seen in third trimester BNT162b2 recipients was highly correlated with FcR-binding and IgG3 titer across SARS-CoV-2 variants, showing that these antibodies are highly inflammatory and likely highly functional. To assess differences in the vaccine-induced immune response transferred from maternal to fetal circulation by vaccine type, we plotted the Spike-specific antibody titer and FcR-binding in umbilical cord serum in the 123 dyads who underwent systems serology profiling. In the cord blood, Spike-specific antibody titers and Fc-receptor binding were significantly higher in recipients of mRNA-1273 or BNT162b2 compared to recipients of Ad26.COV2.S ( Figure 3A and Figure S3A ). IgG2 against Spike was significantly higher in the cord blood of mRNA-1273 recipients compared to either Ad26.COV2.S or BNT162b2 recipients ( Figure S3A ). Moreover, Spike-specific antibody titers and functions in cord blood were highly correlated with the All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint antibody response against variants of concern across all vaccine platforms, suggesting that Spike-specific antibodies in cord blood are likely to be active against variants of concern ( Figure S3B ). These observed differences in Spike-specific antibody response and response to variants of concern by vaccine platform in umbilical cord blood mirrored those observed in the maternal antibody response. Functional antibody responses, including ADCP, ADNP, ADCD, and ADNKA (measured as % CD107a+, % MIP-1β+ and % IFNγ+ cells), were lower in the cord blood of Ad26.COV2.S recipients compared to mRNA-1273 or BNT162b2 ( Figure 3B and S3C). Interestingly, the ADCP and ADCD responses in the cord blood of women who received mRNA-1273 were significantly higher than those of women who received either Ad26.COV2.S or BNT162b2 ( Figure 3B ), whereas the response in the maternal blood was similar between those two vaccines ( Figure 1B and Figure S1B ), suggesting preferential transfer of these highly-functional antibodies in mRNA-1273 recipients. Similar to what was observed in the maternal blood, vaccination with Ad26.COV2.S resulted in equivalent IgG1 titer and FcR-binding against S1 in cord blood compared to mRNA vaccination, but significantly lower FcR-binding antibodies (FcR2a, FcR2b, and FcR3a) against S2 in cord blood ( Figure 3C) . A LASSO-PLSDA model was built using antibody features in cord blood to elucidate which antibody classes are enriched in the cord blood across vaccine platforms ( Figure 3D ). Whereas the mRNA-1273 and BNT162b2 cord blood responses had significant overlap, the Ad26.COV2.S cord blood response separated from the two other vaccine responses ( Figure 3D ). Moreover, all LASSO-selected features were enriched in the cord blood of women who had received mRNA-1273 or BNT162b2 compared to Ad26.COV2.S ( Figure 3E ). These data demonstrate strong similarities between maternal and cord blood antibody titers and functions, All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint and reduced titer, FcR-binding and functionality of cord blood antibodies in recipients of Ad26.COV2.S relative to the mRNA vaccines. To further probe the contribution of vaccine type on transplacental antibody transfer, we plotted the matched maternal-cord antibody titers and functions for each vaccine and compared differences ( Figure 4A -B). While we expect titers to be higher in the cord relative to maternal serum for most vaccine-induced antibodies, [33] [34] [35] it was notable that no antibody feature was significantly higher in the cord blood of women who had received the Ad26.COV2.S vaccine ( Figure 4A -B). In contrast, nearly all Spike-specific antibody functions were higher in cord blood of women who received mRNA-1273 or BNT162b2 compared to maternal blood (ADCP, ADNP, and ADNKA by %CD107a+), with the exception of ADCD which was significantly lower in the cord relative to maternal blood of BNT162b2 recipients ( Figure 4B ). Given that transplacental transfer of antibody is driven substantially by maternal titers, 36, 37 it is possible that the lower transfer of antibodies in women that received Ad26.COV2.S could simply be due to lower maternal titers after Ad26.COV2.S compared to the mRNA vaccines. To reveal whether different vaccine platforms result in an enrichment of different antibody features in the cord blood, we performed a multilevel PLSDA (mPLSDA) using LASSO to select features that were most different between maternal and cord blood for each vaccine ( Figure 4C -E, Figure S4 ). This approach accounts for the heterogeneous responses between vaccine recipients at the individual level. All three vaccines showed separation between maternal and cord blood and an enrichment of FcR-binding and Spike-specific IgG titer in the cord blood relative to maternal ( Figure 4C -E, Figure S4 ). Interestingly, while we did not observe any significant differences between maternal and cord blood in Ad26.COV2.S vaccine recipients through univariate analysis, on a multivariate level, FcR2-binding antibodies, anti-S1 IgG2, and NK-cell-activating antibodies (MIP-1β) were enriched in cord blood of the Ad26.COV.S dyads, while maternal All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint blood was enriched for anti-S2 IgG1 and IgG3, and ADCD ( Figure 4C , Figure S4A ). Thus, despite lower efficiency of antibody transfer in women that received Ad26.COV2.S, all three vaccines allowed for preferential transfer of specific antibodies to the cord blood. Similarly, the cord blood of mRNA-1273 or BNT162b2 recipients was enriched for IgG1 and IgG2, FcRbinding antibodies, functional antibodies, and antibodies directed against variants of concern (Alpha, Beta, and Delta), whereas maternal blood was enriched for IgG3 ( Figure 4D -E, Figure S4B -C). These data highlight that although slight differences in transfer efficiency exist between vaccines, placental enrichment for highly functional antibody in the umbilical cord is a commonality that likely reflects a fundamental principle of transplacental transfer biology. To investigate the impact of trimester of vaccination on the transplacental transfer of vaccineinduced immunity to the neonate at delivery, we measured total anti-Spike antibody IgG (as assessed by ELISA, see Methods) in the 123 dyads included in systems serology analyses and an additional 52 dyads who had delivered by study completion (N=175 total dyads, Supplemental Table 3 ). Interestingly, anti-Spike antibody titers in umbilical cord blood were higher than maternal titers at delivery when vaccination occurred in the first and second but not third trimesters ( Figure 5A ). The transfer ratio (TR)defined as the ratio of cord blood anti-Spike IgG titer to maternal anti-Spike IgG titer at deliverywas calculated for each maternal-neonatal dyad and plotted by trimester of vaccination as a metric of transfer efficiency ( Figure 5B ). This analysis revealed higher TRs generated by first and second trimester vaccination (median TR = 1.5 and 1.3) compared to third trimester vaccination (median TR = 1.0). For reference, the expected efficiency of transplacental antibody transfer is > 1, indicating higher cord titers at delivery All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint compared to maternal titers, with TRs of 1.2-3 at delivery noted for other vaccine-induced titers, such as measles, influenza, and pertussis. [33] [34] [35] We next sought to determine absolute anti-Spike IgG titer in the cord blood at delivery by trimester of vaccination. Total anti-Spike IgG after first trimester vaccination was significantly lower than that in the cord blood of second trimester vaccine recipients ( Figure 5C ). In the context of the highest TRs observed in first trimester vaccine recipients, this finding likely reflects a waning of maternal titers at delivery compared to second trimester vaccine recipients. Given the finding of highest TRs for first trimester vaccination but lower absolute titers in cords of mothers vaccinated in the first trimester, suggestive of high placental efficiency in the setting of maternal first trimester vaccination but waning maternal titers by delivery, we next sought to quantify the waning of maternal antibody titers in first and second trimester vaccine recipients from completion of vaccination to delivery. In the subset of dyads in whom blood was drawn at 2-6 weeks following second vaccine dose in mRNA vaccine recipients or following the single Ad26.COV2.S dose, and again at delivery (n=7 first trimester, n = 19 second trimester), we compared total maternal anti-Spike IgG post-boost (second dose) and at delivery. This analysis demonstrated that antibody titers were significantly lower at delivery compared to shortly after the boost dose in both first and second trimester vaccine recipients ( Figure 5D ). As expected, the ratio of titers at delivery to post-boost was lower for first trimester vaccine recipients compared to second trimester vaccine recipients ( Figure 5E ), likely reflecting a more significant waning of antibody titer with time since vaccination. Pregnancy is a unique immunological epoch, requiring complex and trimester-specific alterations in the maternal immune response to both protect the maternal-neonatal dyad and All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint promote maternal tolerance of the semi-foreign fetal allograft. [22] [23] [24] The COVID-19 pandemic revealed key deficits in our knowledge of both normal and challenged pregnancy immunity as well as the maternal response to vaccines. Although many vaccines can be safely administered in pregnancy, with seasonal influenza and tetanus-diphtheria-acellular pertussis (Tdap) routinely recommended for all pregnant individuals, 21 indicating comparatively less functionality against variants of concern in second trimester versus third trimester vaccination. Although total spike-specific antibody titers were lower in the umbilical cord at delivery following first trimester vaccination, we observed the most efficient transfer of highly functional antibodies from mother to umbilical cord following first trimester vaccination, likely reflective of the placenta working to preserve protection for the newborn in the setting of waning maternal titers over time. For unvaccinated individuals who become pregnant, receiving COVID-19 vaccination in the first trimester may therefore optimize benefit for mother and fetus by protecting against hospitalization, severe morbidity, and death due to COVID-19 over the greatest amount of time during pregnancy, without significantly compromising neonatal immune protection. Both maternal and neonatal immunity can be further enhanced by first trimester maternal vaccination, followed by boosting in the third trimester, with boosting 6 months post-mRNA vaccines and two months post-Ad26.COV2.S vaccine now recommended All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Ad26.COV2.S vaccine recipients and mRNA vaccine recipients might be a reflection of a oneversus two-dose regimen, rather than a reflection of an inferior response to the Ad-vectored vaccine platform itself. This concept is supported by recent data suggesting that the Ad26.COV2.S vaccine protection is enhanced in a two-dose regimen, 51,52 with "booster" dose recommended any time two months or more after initial dose to enhance protection in specific vulnerable populations. 43 Further evaluation of whether the differences seen between mRNA vaccines and the Ad26.COV2.S vaccine persist after two doses of the Ad-vectored vaccine is a critical area for future research, and will elucidate whether the differences noted in pregnancy relate simply to dosing and interval, versus the Ad-vectored platform itself. As previous work has All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. shown the importance of adherence to the prime/boost timeline for mRNA vaccine recipients given delayed kinetics of antibody responses during pregnancy, 13 investigating the impact of "booster" doses given during pregnancy, particularly in recipients who originally received the Ad26.COV2.S vaccine, will be important to obtaining a full understanding of how vaccine strategies can be tailored to optimize maternal protection. Robust changes in the inflammatory profile occur during pregnancy to facilitate implantation and early placentation, followed by a period of rapid fetal growth, and finally, the onset of parturition. [22] [23] [24] [25] How these immune fluctuations influence maternal responses to vaccines administered across gestation is not known. Although univariate analyses examining responses by trimester did not reveal trimester-specific differences, these analyses likely fail to account for interactions between multiple elements of the antibody response. Harnessing the strength of the systems serology approach, we identified superior immunogenicityas characterized by anti-Spike antibody FcR-binding capacity and functionalityin pregnant individuals vaccinated in the first and third trimesters compared to those vaccinated in the second trimester through multivariate modeling. These data demonstrate the importance of considering more than just Ig titer alone when evaluating vaccine immunogenicity. Further investigation into the anti-Spike antibody responses (including against variants of concern) in second and third trimester vaccine recipients by vaccine type revealed that second trimester responses are impaired compared with third trimester for both mRNA vaccines. Taken together, these data suggest that second trimester vaccination generates an antibody response characterized by overall reduced FcRbinding capacity and functionality relative to vaccination in the first and third trimesters. These findings can be understood in the context of immunomodulatory changes that occur in the second trimester of pregnancy that favor maternal tolerance of the developing fetal semiallograft and promote a state of immunological quiescence, 25 in which response to non-self antigens may be dampened. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint While effective maternal protection is paramount during a pandemic, neonatal protection against potentially harmful pathogens through maternal immunization is an important secondary consideration when developing vaccine recommendations in pregnancy. 20,21 Recipients of all 3 COVID-19 vaccines demonstrated enrichment of FcR-binding and anti-Spike IgG titer in the cord compared with maternal blood, with the most favorable cord:maternal transfer ratios (greater than 1) following first and second trimester vaccination. Whether higher antibody transfer ratios observed for first trimester COVID-19 vaccination are due to differences in antibody Fc-quality and thus affinity for Fc-receptors that traffic antibody to the fetal circulation, 37, 53, 54 or are the result of increased time for antibody transit to occur, or a combination of both is yet to be determined. As expected, the waning of maternal titers was more significant after first compared to second trimester maternal vaccination, and both maternal and neonatal immunity would therefore be boosted by maternal COVID-19 boosting in the third trimester, when initial vaccination (or vaccination series) occurs in the first trimester. These results add significantly to our current understanding of how timing of maternal vaccination impacts both maternal immune response and transplacental transfer efficiency. Current clinical recommendations governing the timing of routinely administered vaccines in pregnancy (e.g. influenza vaccine, which is administered during influenza season regardless of trimester, and Tdap, which is administered in late second to early third trimester with the primary goal of enhancing transplacental transfer) have limited the ability to systematically investigate the impact of vaccine administration across gestation. The Advisory Committee on Immunization Practices (ACIP) has advised routine Tdap administration during pregnancy after 20 weeks of gestation for over a decade, based on limited availability of safety data in the first trimester. 55 Studies that have investigated the timing of Tdap vaccination after the first trimester have found superior transfer of anti-pertussis antibody when vaccination occurs earlier in the All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint recommended interval of 27-36 weeks, 41,56,57 with some evidence from individuals vaccinated outside that window favoring improved transfer following early second trimester vaccination. 58 Data on placental transfer of anti-pertussis antibody following Tdap administration in the first trimester are not available, as studies including first trimester vaccinees are primarily limited to safety reports. 59,60 Data from seasonal influenza vaccine administration in pregnancy, which is administered at any gestational age during influenza season, are conflicting with respect to maternal immune response. Some studies suggest lower maternal anti-influenza titers in first compared to second trimester vaccination, with highest anti-influenza titers in third trimester vaccination, 42,61,62 while others suggest a more robust maternal titer generated by first and third trimester vaccination relative to second. 63 In addition, a majority of studies noted enhanced cord blood antibody titers against influenza following third trimester vaccination when compared with second or first trimester vaccination, likely due in part to waning maternal antibody titers with increased time from vaccination. 42 These studies were limited by their narrow focus on IgG titer as the primary measure of maternal immune response, while our systems serology approach permits the dissection of diverse components of maternal humoral immunity. Whether differences in cord blood titers observed between first, second, and third trimester vaccination correlate with differential neonatal protection from COVID-19 will be important to assess in neonatal cohorts. The rapid development and distribution of three novel COVID-19 vaccines in the US has offered an unprecedented opportunity to further our understanding of the rules of vaccine-induced immunity in pregnancy. Our study contributes substantially to knowledge of how the maternalneonatal dyad responds to vaccination against a de novo antigen with novel mRNA and Advectored vaccines, which were not specifically designed to optimize maternal or neonatal protection, as pregnant individuals were excluded from initial vaccine clinical trials. [9] [10] [11] Looking beyond responses to the COVID-19 vaccines, our findings may have broader implications. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint These insights into both platform-and trimester-specific differences to COVID-19 vaccines can be used to guide rational vaccine development and enhance understanding of the maternal immune response to varied perturbations across trimesters. Optimizing both maternal and neonatal immunity is a key consideration informing vaccination strategies in this unique population, and our data suggest that for the COVID-19 vaccines, maternal immunization in the first trimester may optimize benefit to both mother and neonate, particularly if boosting is employed in the third trimester. Efforts to recruit and include pregnant individuals in vaccine studies will remain critical to constructing evidence-based vaccine strategies that maximize benefit to both mother and newborn. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (E) The barplot shows the latent variable (LV) 1 for the least absolute shrinkage and selection operator (LASSO)-selected features for the PLSDA in (D). Features that with a positive LV1 loading were enriched in the cords whose mothers received an mRNA vaccine. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint B. The dot plot shows the transfer ratio (cord titer/maternal titer) of Spike-specific IgG to the cord. Color indicates trimester of vaccination. Significance was determined by Kruskal-Wallis test. **** p < 0.0001. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint C. The dot plot shows the Spike-specific IgG titer in cord blood by trimester that the mother received COVID-19 vaccination. Significance was determined by a one-way ANOVA followed by correction for multiple comparisons, * p < 0.05. D. The dot plots show the IgG Spike titer in maternal plasma post-boost (~2-6 weeks after second dose of mRNA vaccine or after single dose of Ad26.COV2.S vaccine) and at delivery. Lines connect matched samples. Significance was determined by a Wilcoxon signed rank test, * p < 0.05, ** p < 0.01. E. The dot plot shows the ratio of the IgG Spike titer delivery/post-boost. Significance was determined by Mann Whitney test, ** p < 0.01. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint Pregnant individuals at two tertiary care centers were approached for enrollment in the COVID- For Ad26.COV2.S vaccine recipients, blood was collected at least 2 weeks after receiving the single vaccine dose. For mRNA-1273 and BNT162b2 vaccine recipients, blood was collected at least 2 weeks following the second vaccine dose. For participants who delivered during the study time frame (N=123), maternal blood was drawn at the time of delivery, and umbilical cord blood was collected after delivery. Blood was collected by venipuncture (or from the umbilical vein following delivery) into serum separator and EDTA tubes and centrifuged at 1000 g for 10 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint min at room temperature. Serum and plasma were aliquoted into cryogenic vials and stored at -80°C. Antigens used for assays included SARS-CoV-2 D614G Spike, Alpha Spike, Beta Spike, Gamma Spike and Delta Spike (all Spikes kindly provided by Erica Ollman Saphire) and SARS-CoV-2 S1 and S2 (Sino Biological). Neutrophils were isolated from fresh peripheral whole blood collected at the Ragon Institute. NK cells were isolated from fresh peripheral blood from buffy coats collected at Massachusetts General Hospital (MGH). All volunteers gave signed, informed consent and were over the age of 18, and samples were deidentified before use. The study was approved by the MGH Institutional Review Board. Neutrophils were maintained in R10 media (RPMI supplemented 10% fetal bovine serum (FBS) (Sigma Aldrich), 5% penicillin/streptomycin (Corning, 50 µg/mL), 5% L-glutamine (Corning, 4 mM), 5% HEPES buffer (pH 7.2) (Corning, 50 mM)) and at 37°C, 5% CO2 for the duration of the assay. After isolation, NK cells were rested overnight at R10 media supplemented with 2ng/mL interleukin (IL)-15 at 37°C, 5% CO2. For antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP) and antibody-dependent complement deposition (ADCD), D614G Spike was biotinylated using Sulfo-NHS-LC-LC biotin (Thermo Fisher Scientific) and desalted using Zeba Columns (Thermo Fisher Scientific). Biotinylated antigen was coupled to yellow-green FluoSpheres NeutrAvidin beads (for ADCP and ADNP) or red neutravidin beads (for ADCD) (Invitrogen). To form immune complexes, antigen-coupled beads were incubated with All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint appropriately diluted serum (1:100 for ADCP, 1:50 for ADNP and 1:10 for ADCD) for 2 hours at 37°C. Immune complexes were then washed. For ADCP, THP-1 cells (ATCC) were added to plates at a concentration of 2.5x10 4 cells/mL. Cells were incubated for 16-18 hours at 37°C with the immune complexes and fixed following the incubation. Fluorescence was acquired using an iQue (Intellicyt). For ADNP, leukocytes were isolated from fresh peripheral blood using ACK Lysing Buffer (Thermo Fisher Scientific). Leukocytes were added at a concentration of 5x10 4 cells/mL. Cells were incubated for 1 hour at 37°C with the immune complexes. Following the incubation, neutrophils were stained using anti-CD66b Pacblue (biolegend). Cells were then fixed. Fluorescence of CD66b+ cells was acquired using an iQue (Intellicyt). For ADCP and ADNP, a phago score was calculated using the following formula: (% fluorescent cells*MFI of fluorescent cells)/10000. For ADCD, lyophilized guinea pig complement (Cedarlane) was diluted in gelatin veronal buffer supplemented with calcium and magnesium. The diluted guinea pig complement was added to immune complexes and plates were incubated at 37°C for 20 minutes. Plates were washed with 15 µM EDTA diluted in PBS. Complement was stained using anti-C3 FITC (MP BioProducts). Fluorescence was determined using an iQue (Intellicyt). For all functional assays, samples were run in duplicate and data is reported as the average of the replicates. ELISA plates were coated with 2 µg/mL of Spike protein. Plates were washed and blocked with 5% BSA in PBS. Immune complexes were formed by adding serum diluted 1:25 to plates and incubating plates for 2 hours at 37°C. RosetteSep (STEMCELL Technologies) and a ficoll gradient was used to isolate NK cells from fresh peripheral blood from healthy donors. Isolated NK cells were rested overnight in R10 (see Primary Cells section above) with 2 ng/mL of IL-15. NK cells were added to immune complexes at a concentration of 5 x 10 4 cells/mL in media supplemented with Brefeldin A (Sigma), anti-CD107a BV605 (BD Biosciences) and GolgiStop All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint (BD Biosciences). NK cells were incubated with immune complexes for 5 hours at 37°C. After incubation, cells were stained for surface markers using anti-CD56 PE-Cy7 (BD Biosciences) and anti-CD3 APC-Cy7 (BD Biosciences). Cells were fixed with PermA (Life Technologies), Permeabilized with Perm B (Life Technologies), and stained with anti-MIP1b-BV421 (BD Biosciences) and anti-IFNg-PE (BD Biosciences). The cells were analyzed for fluorescence using an iQue (Intellicyt). NK cells were gates as CD3-/CD56+ and NK activity was determined as percent of cells positive for CD107a, IFN-g or MIP-1b. A multiplexed Luminex assay was used to determine the relative concentration of antigenspecific antibody isotype and subclass titer and Fc receptor binding. Carboxylated microsphere were coupled to antigen using EDC and Sulfo-NHS (Thermo Fisher Scientific) to form covalent NHS-ester linkages. To form immune complexes, diluted serum (1:100 for IgG2/3, 1:500 for IgG1, and 1:1000 for FcRs) was mixed with antigen-couple microspheres and incubated overnight at 4°C shaking at 700 rpm. The following day, plates were washed three times with 0.1% BSA 0.02% Tween-20 in PBS. Antigen-specific antibody isotypes were measured using PE-coupled mouse anti-human antibodies (Southern Biotech) specific for each specific isotype. Avi-tagged FcRs (Duke Human Vaccine Institute) were biotinylated using a BirA500 kit (Avidity) and tagged with streptavidin-PE. PE-tagged FcRs were added to immune complexes to determine antigen-specific FcR binding. Fluorescence was acquired using an iQue (Intellicyt) and antigen-specific antibody titer and FcR-binding is reported as Median Fluorescence Intensity (MFI). To further assess cord:maternal transfer ratios by trimester of vaccination, maternal and umbilical cord blood samples were collected from an additional 52 participants who delivered All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint during the study period (N=175 total maternal-neonatal dyads included for this analysis). Antibodies against the SARS-CoV-2 Spike were quantified using an ELISA. ELISA plates were coated with 500 ng/mL of D614G Spike (kindly provided by Erica Saphire) and incubated for 30 minutes at room temperature. Plates were washed with washing buffer (0.05% Tween-20. 400mM NaCl, 50mM Tris, pH 8.0) and blocked with a 0.1% BSA solution for 30 minutes at room temperature. Plates were washed, and sample was added at a dilution of 1:100. Plates were incubated with sample at 37°C for 30 minutes. Plates were washed, and a horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody (Bethyl Laboratories) was added for detection of Spike-specific IgG. Plates were incubated with secondary antibody for 30 minutes at room temperature and then washed. TMB was used to develop the ELISA and sulfuric acid was used to stop the ELISA. Signal was read at 450 nm and background corrected from a reference wavelength of 570 nm. For univariate analysis, statistics were calculated using GraphPad Prism version 8.0. Luminex data and ADCD were log10-transformed before analysis. For analysis of differences between vaccines or trimesters, significance was determined by a one-way ANOVA followed by posthoc Benjamini-Hochberg adjustment. For analysis of maternal-cord differences, significance was determined by a Wilcoxon matched pairs signed rank test followed by posthoc Benjamini-Hochberg adjustment. Multivariate analysis was performed in R (version 4.0.0). Prior to building the models, data was centered and scaled. LASSO feature selection was performed using the "select_lasso" function in systemseRology R package (v1.0) (https://github.com/LoosC/systemsseRology) to determine significant features. The LASSO tuning parameter was determined by 5-fold cross validation. LASSO feature selection was performed 100 times, and features that were chosen 50% of the repetitions were selected to build the model. LASSO-selected features were used to build partial least squares discriminant All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint analysis (PLSDA) or multilevel PLSDA models. Model performance was determined by 5-fold cross validation. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266273 doi: medRxiv preprint Update: Characteristics of Symptomatic Women of Reproductive Age with Laboratory-Confirmed SARS-CoV-2 Infection by Pregnancy Status -United States Maternal and Neonatal Morbidity and Mortality Among Pregnant Women With and Without COVID-19 Infection: The INTERCOVID Multinational Cohort Study The Differences in Clinical Presentation, Management, and Prognosis of Laboratory-Confirmed COVID-19 between Pregnant and Non-Pregnant Women: A Systematic Review and Meta-Analysis Characteristics and Maternal and Birth Outcomes of Hospitalized Pregnant Women with Laboratory-Confirmed COVID-19 -COVID-NET, 13 States Association of SARS-CoV-2 Test Status and Pregnancy Outcomes Birth and Infant Outcomes Following Laboratory-Confirmed SARS Vaccine Safety Datalink*, United States Protection by Exclusion: Another Missed Opportunity to Include Pregnant Women in Research During the Coronavirus Disease Involving Pregnant Individuals in Clinical Research on COVID-19 Vaccines Exclusion of pregnant and lactating women from COVID-19 vaccine trials: a missed opportunity COVID-19 vaccine response in pregnant and lactating women: a cohort study COVID-19 mRNA vaccines drive differential antibody Fc-functional profiles in pregnant, lactating, and non-pregnant women Immunogenicity of COVID-19 mRNA Vaccines in Pregnant and Lactating Women Preliminary Findings of mRNA Covid-19 Vaccine Safety in Pregnant Persons Messenger RNA Vaccination in Pregnant Women and Transplacental Passage Into Cord Blood All rights reserved. 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No reuse allowed without permission preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted