SARS-CoV-2 infection elucidates unique features of pregnancy-specific immunity

Pregnancy is a risk factor for increased severity of SARS-CoV-2 and other respiratory infections. The mechanisms underlying this risk have not been well-established, partly due to a limited understanding of how pregnancy shapes immune responses. To gain insight into the role of pregnancy in modulating immune responses at steady state and upon perturbation, we collected peripheral blood mononuclear cells (PBMC), plasma, and stool from 226 women, including 152 pregnant individuals (n = 96 with SARS-CoV-2 infection and n = 56 healthy controls) and 74 non-pregnant women (n = 55 with SARS-CoV-2 and n = 19 healthy controls). We found that SARS-CoV-2 infection was associated with altered T cell responses in pregnant compared to non-pregnant women. Differences included a lower percentage of memory T cells, a distinct clonal expansion of CD4-expressing CD8+T cells, and the enhanced expression of T cell exhaustion markers, such as programmed cell death-1 (PD-1) and T cell immunoglobulin and mucin domain-3 (Tim-3), in pregnant women. We identified additional evidence of immune dysfunction in severely and critically ill pregnant women, including a lack of expected elevation in regulatory T cell (Treg) levels, diminished interferon responses, and profound suppression of monocyte function. Consistent with earlier data, we found maternal obesity was also associated with altered immune responses to SARS-CoV-2 infection, including enhanced production of inflammatory cytokines by T cells. Certain gut bacterial species were altered in pregnancy and upon SARS-CoV-2 infection in pregnant individuals compared to non-pregnant women. Shifts in cytokine and chemokine levels were also identified in the sera of pregnant individuals, most notably a robust increase of interleukin-27 (IL-27), a cytokine known to drive T cell exhaustion, in the pregnant uninfected control group compared to all non-pregnant groups. IL-27 levels were also significantly higher in uninfected pregnant controls compared to pregnant SARS-CoV-2-infected individuals. Using two different preclinical mouse models of inflammation-induced fetal demise and respiratory influenza viral infection, we found that enhanced IL-27 protects developing fetuses from maternal inflammation but renders adult female mice vulnerable to viral infection. These combined findings from human and murine studies reveal nuanced pregnancy-associated immune responses, suggesting mechanisms underlying the increased susceptibility of pregnant individuals to viral respiratory infections. ### Competing Interest Statement A-C.V. has a financial interest in 10X Genomics. The company designs and manufactures gene sequencing technology for use in research, and such technology is being used in this research. A-C.V.'s interests were reviewed by The Massachusetts General Hospital and Mass General Brigham in accordance with their institutional policies. J.R.H. consults for CJ CheilJedang and Interon Laboratories. A.G.E. consults for Mirvie, Inc. outside of this work, and receives research funding from Merck Pharmaceuticals outside of this work. K.J.G has served as a consultant for BillionToOne, Aetion, Roche, and Janssen Global Services outside of this work. ### Funding Statement D.S.O. was supported by a postdoctoral fellowship program (Nurturing Next-generation Researchers) through the National Research Foundation of Korea (2021R1A6A3A14044062). E.K was supported by the National Research Foundation of Korea (RS-2023-00209464) and a Korea University grant (K2225821). R.N. was supported by a postdoctoral fellowship (MGH Executive Committee on Research Medical Discovery (FMD) Fundamental Research Fellowship Award). M.B.G., M.R.F., and N.H. were supported by an American Lung Association COVID-19 Action Initiative grant. M.B.G. and M.R.F. were supported by a grant from the Executive Committee on Research at MGH. P.S. was supported by the NHLBI K08HL157725, an American Heart Association Career Development Award, and the Brigham and Women's Hospital Innovation Evergreen Fund. O.P. and A.L. were supported by the COVID-Relief research funds from the Brigham and Women's Hospital, Department of Psychiatry. A.-C.V. acknowledges funding support from the COVID-19 Clinical Trials Pilot grant from the Executive Committee on Research at MGH, a COVID-19 Chan Zuckerberg Initiative grant (2020-216954), the funds from the Manton Foundation and the Klarman Family Foundation, and the National Institute of Health (DP2CA247831). L.L.S. was supported by the NICHD K12HD103096. K.J.G. was supported by the NHLBI K08 HL146963, K08 HL146963-02S1. This work was also supported by R01HD100022-S2 (to A.G.E.), RF1MH132336 (to A.G.E. and R.H.P.) Simons Foundation SFARI Maternal COVID-19 Award 870754 (to A.G.E. and K.J.G.). J.R.H. was supported by the Jeongho Kim Neurodevelopmental Research Fund, the Simons Foundation Autism Research Initiative, and the Burroughs Wellcome Fund (the Pathogenesis of Infectious Disease Program). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee/IRB of Mass General Brigham gave approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes scRNA data, CITE-seq, and repertoire data were deposited to GEO and will be available in the accession number GSE239452 upon publication. The code used for single-cell analysis and figures generation will be available in GitHub at https://github.com/villani-lab/COVID\_pregnancy\_and in the Zenodo repository upon publication of this study.