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• W2592337312 abstract "•Pregnancy-associated influenza susceptibility can be mimicked in pregnant mice•Allogenically pregnant mice show a more severe infection than do syngenically mated mice•CD8+ T cell migration into the lung is impaired in allogenically pregnant infected mice•H1N1 virus variants emerge in pregnant mice that are more virulent in non-pregnant mice Pregnant women are at high risk for severe influenza disease outcomes, yet insights into the underlying mechanisms are limited. Here, we present models of H1N1 infection in syngenic and allogenic pregnant mice; infection in the latter mirrors the severe course of 2009 pandemic influenza in pregnant women. We found that the anti-viral immune response in the pregnant host was significantly restricted as compared to the non-pregnant host. This included a reduced type I interferon response as well as impaired migration of CD8+ T cells into the lung. The multi-faceted failure to mount an anti-viral response in allogenic pregnant mice resulted in a less stringent selective environment that promoted the emergence of 2009 H1N1 virus variants that specifically counteract type I interferon response and mediate increased viral pathogenicity. These insights underscore the importance of influenza vaccination compliance in pregnant women and may open novel therapeutic avenues. Pregnant women are at high risk for severe influenza disease outcomes, yet insights into the underlying mechanisms are limited. Here, we present models of H1N1 infection in syngenic and allogenic pregnant mice; infection in the latter mirrors the severe course of 2009 pandemic influenza in pregnant women. We found that the anti-viral immune response in the pregnant host was significantly restricted as compared to the non-pregnant host. This included a reduced type I interferon response as well as impaired migration of CD8+ T cells into the lung. The multi-faceted failure to mount an anti-viral response in allogenic pregnant mice resulted in a less stringent selective environment that promoted the emergence of 2009 H1N1 virus variants that specifically counteract type I interferon response and mediate increased viral pathogenicity. These insights underscore the importance of influenza vaccination compliance in pregnant women and may open novel therapeutic avenues. Influenza severely affects human populations through seasonal epidemics and random pandemics. While influenza epidemics occur yearly during autumn and winter (Jamieson et al., 2009Jamieson D.J. Honein M.A. Rasmussen S.A. Williams J.L. Swerdlow D.L. Biggerstaff M.S. Lindstrom S. Louie J.K. Christ C.M. Bohm S.R. et al.Novel Influenza A (H1N1) Pregnancy Working GroupH1N1 2009 influenza virus infection during pregnancy in the USA.Lancet. 2009; 374: 451-458Abstract Full Text Full Text PDF PubMed Scopus (1137) Google Scholar), influenza pandemics strike irregularly, but recurrently. The most recent influenza pandemic occurred in the year 2009, when a new H1N1 influenza virus strain emerged (Dawood et al., 2012Dawood F.S. Iuliano A.D. Reed C. Meltzer M.I. Shay D.K. Cheng P.Y. Bandaranayake D. Breiman R.F. Brooks W.A. Buchy P. et al.Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study.Lancet Infect. Dis. 2012; 12: 687-695Abstract Full Text Full Text PDF PubMed Scopus (864) Google Scholar, Garten et al., 2009Garten R.J. Davis C.T. Russell C.A. Shu B. Lindstrom S. Balish A. Sessions W.M. Xu X. Skepner E. Deyde V. et al.Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans.Science. 2009; 325: 197-201Crossref PubMed Scopus (2003) Google Scholar). One common denominator of all influenza pandemics recorded over the last century is the proportionally high morbidity and mortality rate among pregnant women (Freeman and Barno, 1959Freeman D.W. Barno A. Deaths from Asian influenza associated with pregnancy.Am. J. Obstet. Gynecol. 1959; 78: 1172-1175Abstract Full Text PDF PubMed Scopus (293) Google Scholar, Jamieson et al., 2009Jamieson D.J. Honein M.A. Rasmussen S.A. Williams J.L. Swerdlow D.L. Biggerstaff M.S. Lindstrom S. Louie J.K. Christ C.M. Bohm S.R. et al.Novel Influenza A (H1N1) Pregnancy Working GroupH1N1 2009 influenza virus infection during pregnancy in the USA.Lancet. 2009; 374: 451-458Abstract Full Text Full Text PDF PubMed Scopus (1137) Google Scholar). Pregnancy creates a unique immunological situation in allogenic matings, since the placenta—which is in direct contact with the maternal immune system—expresses antigens derived from the father. To tolerate this “foreign” allogenic tissue, the maternal immune system in mice and humans mounts intricate processes of adaptation, which ensure that rejection of the fetus is suppressed (Arck and Hecher, 2013Arck P.C. Hecher K. Fetomaternal immune cross-talk and its consequences for maternal and offspring’s health.Nat. Med. 2013; 19: 548-556Crossref PubMed Scopus (406) Google Scholar). These adaptational processes include the arrest of dendritic cells (DCs) in a tolerogenic state, mirrored by a reduced expression of costimulatory surface markers (Blois et al., 2007Blois S.M. Ilarregui J.M. Tometten M. Garcia M. Orsal A.S. Cordo-Russo R. Toscano M.A. Bianco G.A. Kobelt P. Handjiski B. et al.A pivotal role for galectin-1 in fetomaternal tolerance.Nat. Med. 2007; 13: 1450-1457Crossref PubMed Scopus (387) Google Scholar, Segerer et al., 2008Segerer S.E. Müller N. Brandt Jv. Kapp M. Dietl J. Reichardt H.M. Rieger L. Kämmerer U. The glycoprotein-hormones activin A and inhibin A interfere with dendritic cell maturation.Reprod. Biol. Endocrinol. 2008; 6: 17Crossref PubMed Scopus (40) Google Scholar). Moreover, effector T cells are constrained in their migration to the feto-maternal interface due to the epigenetic silencing of the C-X-C motif chemokine (CXCL) 10, a chemoattractant involved in recruiting CXCR3+ leukocytes, into the uterus (Nancy et al., 2012Nancy P. Tagliani E. Tay C.S. Asp P. Levy D.E. Erlebacher A. Chemokine gene silencing in decidual stromal cells limits T cell access to the maternal-fetal interface.Science. 2012; 336: 1317-1321Crossref PubMed Scopus (288) Google Scholar, Chaturvedi et al., 2015Chaturvedi V. Ertelt J.M. Jiang T.T. Kinder J.M. Xin L. Owens K.J. Jones H.N. Way S.S. CXCR3 blockade protects against Listeria monocytogenes infection-induced fetal wastage.J. Clin. Invest. 2015; 125: 1713-1725Crossref PubMed Scopus (57) Google Scholar). Tolerance toward the fetus and pregnancy maintenance is further sustained by the generation of CD4+ and CD8+ regulatory T (Treg), and Natural Killer (NK) cells that reside at the feto-maternal interface (Arck and Hecher, 2013Arck P.C. Hecher K. Fetomaternal immune cross-talk and its consequences for maternal and offspring’s health.Nat. Med. 2013; 19: 548-556Crossref PubMed Scopus (406) Google Scholar, Solano et al., 2015Solano M.E. Kowal M.K. O’Rourke G.E. Horst A.K. Modest K. Plösch T. Barikbin R. Remus C.C. Berger R.G. Jago C. et al.Progesterone and HMOX-1 promote fetal growth by CD8+ T cell modulation.J. Clin. Invest. 2015; 125: 1726-1738Crossref PubMed Scopus (59) Google Scholar, Moffett and Colucci, 2014Moffett A. Colucci F. Uterine NK cells: active regulators at the maternal-fetal interface.J. Clin. Invest. 2014; 124: 1872-1879Crossref PubMed Scopus (270) Google Scholar). Also, B cells are increasingly recognized to promote fetal tolerance (Muzzio et al., 2014Muzzio D.O. Soldati R. Ehrhardt J. Utpatel K. Evert M. Zenclussen A.C. Zygmunt M. Jensen F. B cell development undergoes profound modifications and adaptations during pregnancy in mice.Biol. Reprod. 2014; 91: 115Crossref PubMed Scopus (72) Google Scholar). These intricate pathways collectively create a tolerogenic, anti-inflammatory environment in which placental development and fetal growth can occur. In contrast to this tolerogenic immune response during pregnancy, influenza virus infection generally leads to an immediate activation of the immune response to mount anti-viral immunity and clear the infection (Gabriel and Arck, 2014Gabriel G. Arck P.C. Sex, immunity and influenza.J. Infect. Dis. 2014; 209: S93-S99Crossref PubMed Scopus (67) Google Scholar, Miller et al., 2015Miller M.A. Ganesan A.P. Luckashenak N. Mendonca M. Eisenlohr L.C. Endogenous antigen processing drives the primary CD4+ T cell response to influenza.Nat. Med. 2015; 21: 1216-1222Crossref PubMed Scopus (59) Google Scholar). Here, the production of anti-viral, inflammatory cytokines such as interferon (IFN)-α/β by epithelial and immune cells is a critical first line of defense. Moreover, DCs become antigen presenting cells and induce the generation of virus-specific effector T cells (Gabriel and Arck, 2014Gabriel G. Arck P.C. Sex, immunity and influenza.J. Infect. Dis. 2014; 209: S93-S99Crossref PubMed Scopus (67) Google Scholar, Unkel et al., 2012Unkel B. Hoegner K. Clausen B.E. Lewe-Schlosser P. Bodner J. Gattenloehner S. Janßen H. Seeger W. Lohmeyer J. Herold S. Alveolar epithelial cells orchestrate DC function in murine viral pneumonia.J. Clin. Invest. 2012; 122: 3652-3664Crossref PubMed Scopus (80) Google Scholar, Lambrecht and Hammad, 2012Lambrecht B.N. Hammad H. Lung dendritic cells in respiratory viral infection and asthma: from protection to immunopathology.Annu. Rev. Immunol. 2012; 30: 243-270Crossref PubMed Scopus (232) Google Scholar). Facilitated by chemokines, virus-specific effector T (Teff) cells subsequently migrate into the lung to clear the infection (Lambrecht and Hammad, 2012Lambrecht B.N. Hammad H. Lung dendritic cells in respiratory viral infection and asthma: from protection to immunopathology.Annu. Rev. Immunol. 2012; 30: 243-270Crossref PubMed Scopus (232) Google Scholar). These differential, if not opposing immune responses mounted during pregnancy or upon influenza virus infection strongly indicate that the pregnant host may face a contradictory demand to sustain immune tolerance required for fetal survival versus mounting an inflammatory response to eliminate the influenza virus (Gabriel and Arck, 2014Gabriel G. Arck P.C. Sex, immunity and influenza.J. Infect. Dis. 2014; 209: S93-S99Crossref PubMed Scopus (67) Google Scholar, Krishnan et al., 2013Krishnan L. Nguyen T. McComb S. From mice to women: the conundrum of immunity to infection during pregnancy.J. Reprod. Immunol. 2013; 97: 62-73Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, Pazos et al., 2012aPazos M. Sperling R.S. Moran T.M. Kraus T.A. The influence of pregnancy on systemic immunity.Immunol. Res. 2012; 54: 254-261Crossref PubMed Scopus (152) Google Scholar). However, insights into the underlying pathways of this presumed predicament of the pregnant host’s immune response are still sparse. Mouse models, which are pivotal tools to understand the immune response to pregnancy as well as to influenza virus infection, may help to identify such pathways. Indeed, some evidence is available to support an altered influenza-related morbidity during pregnancy (Chan et al., 2010Chan K.H. Zhang A.J. To K.K. Chan C.C. Poon V.K. Guo K. Ng F. Zhang Q.W. Leung V.H. Cheung A.N. et al.Wild type and mutant 2009 pandemic influenza A (H1N1) viruses cause more severe disease and higher mortality in pregnant BALB/c mice.PLoS ONE. 2010; 5: e13757Crossref PubMed Scopus (66) Google Scholar, Marcelin et al., 2011Marcelin G. Aldridge J.R. Duan S. Ghoneim H.E. Rehg J. Marjuki H. Boon A.C. McCullers J.A. Webby R.J. Fatal outcome of pandemic H1N1 2009 influenza virus infection is associated with immunopathology and impaired lung repair, not enhanced viral burden, in pregnant mice.J. Virol. 2011; 85: 11208-11219Crossref PubMed Scopus (64) Google Scholar, Kim et al., 2012Kim H.M. Kang Y.M. Song B.M. Kim H.S. Seo S.H. The 2009 pandemic H1N1 influenza virus is more pathogenic in pregnant mice than seasonal H1N1 influenza virus.Viral Immunol. 2012; 25: 402-410Crossref PubMed Scopus (24) Google Scholar). However, these studies were carried out in syngenically mated mice, which limits the translational relevance of the obtained findings for human pathologies. Thus, a tailored assessment of the unique features and facets of maternal immune responses mounted against an allogenic fetus during pregnancy was urgently needed, which prompted us to perform the present study. Infection of BALB/c-mated allogenic pregnant C57BL/6 females at mid-pregnancy with a dose of 103 plaque forming units (PFU) of the 2009 pandemic (pH1N1) influenza virus resulted in an increased mortality in allogenic pregnant mice, compared to pH1N1 infected, C57BL/6-mated, syngenic pregnant C57BL/6 females (Figure 1A). Moreover, while surviving syngenic pregnant mice fully recovered within 14 days post infection (d.p.i.), as determined by a restored body weight as a proxy of morbidity during influenza infection (Figure 1B) (Gabriel et al., 2005Gabriel G. Dauber B. Wolff T. Planz O. Klenk H.D. Stech J. The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host.Proc. Natl. Acad. Sci. USA. 2005; 102: 18590-18595Crossref PubMed Scopus (548) Google Scholar), allogenic pregnant mice showed an increased and prolonged weight loss upon infection (Figure 1C). In non-pregnant, pH1N1 infected C57BL/6 females, mortality rates were not increased upon infection, and full recovery from influenza-related morbidity was reached at 14 d.p.i. (Figures 1D and 1E). Virus titers were higher on day 6 p.i. in the lungs of 2009 pH1N1 infected allogenic compared to syngenic pregnant mice (Figure 1F). Consistently, lungs of infected syngenic and allogenic pregnant mice presented increased numbers of infiltrated mononuclear cells defined as inflamed areas and higher frequencies of viral antigen (NP) positive areas compared to infected non-pregnant mice (Figures 1G–1I). To test if the pregnancy-related mortality and morbidity is specific for the pH1N1 virus, we also infected pregnant and non-pregnant mice with 103 PFU of a 2006 seasonal H1N1 (sH1N1) strain, where fewer influenza-related complications were reported among pregnant women (Rasmussen and Jamieson, 2012Rasmussen S.A. Jamieson D.J. Influenza and pregnancy in the United States: before, during, and after 2009 H1N1.Clin. Obstet. Gynecol. 2012; 55: 487-497Crossref PubMed Scopus (19) Google Scholar). Here, the infection did not cause a significant weight loss or mortality in pregnant or non-pregnant mice (Figures 1J–1L). Similarly, increasing the sH1N1 virus concentration by 100× (105 PFU), all infected pregnant and non-pregnant mice survived. When reducing the pH1N1 virus infection dose 10× (102 PFU), the infection became non-lethal also in allogenic pregnant mice but still mediated a significant weight loss. When using a 10× higher pH1N1 virus dose (104 PFU), mortality and weight loss further increased in pregnant mice, and survival rate also decreased in non-pregnant mice (Figure S1). We further observed that more animals presented systemic viral titers—assessed in the gut (Otte et al., 2011Otte A. Sauter M. Alleva L. Baumgarte S. Klingel K. Gabriel G. Differential host determinants contribute to the pathogenesis of 2009 pandemic H1N1 and human H5N1 influenza A viruses in experimental mouse models.Am. J. Pathol. 2011; 179: 230-239Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar)—in the allogenic pregnant pH1N1-infected groups. However, NP+ cells or viral titers were not detectable in placental tissue or viral titers in concepti. Also, no significant alterations of the course and outcome of pregnancy were present in infected syn- or allogenic pregnant mice (Figure S2). Overall, these findings mirror the clinical observation of pH1N1-specific increase in morbidity and mortality during pregnancy, which is particularly profound in allogenic murine matings. Considering that allogenic matings in mice are also more comparable to human pregnancies, we performed all subsequent experiments exclusively in allogenic matings. The host innate response, particularly type I IFNs, present the first line of defense against viral pathogens (Schneider et al., 2014Schneider W.M. Chevillotte M.D. Rice C.M. Interferon-stimulated genes: a complex web of host defenses.Annu. Rev. Immunol. 2014; 32: 513-545Crossref PubMed Scopus (1728) Google Scholar). Thus, we investigate the expression pattern of key innate response genes during pregnancy by performing an mRNA expression analysis using next-generation sequencing (RNA-seq) of infected and uninfected lungs in pregnant versus non-pregnant mice. The mRNA transcripts were further analyzed using an innate immune response database, which covers greater than 196,000 experimentally validated molecular interactions within the inflammatory host response (Breuer et al., 2013Breuer K. Foroushani A.K. Laird M.R. Chen C. Sribnaia A. Lo R. Winsor G.L. Hancock R.E. Brinkman F.S. Lynn D.J. InnateDB: systems biology of innate immunity and beyond--recent updates and continuing curation.Nucleic Acids Res. 2013; 41: D1228-D1233Crossref PubMed Scopus (702) Google Scholar). We found numerous genes, which were differentially regulated in infected pregnant versus non-pregnant lungs (Table S1). Most importantly, a clear pattern of downregulated innate response genes was present in 2009 pH1N1 infected pregnant compared to non-pregnant mice on day 6 p.i. (Figure 2A). We then re-assessed a number of differentially downregulated innate factors, such as IFN-γ, interleukin (IL)-6, and tumor necrosis factor (TNF)-α, in the lungs of infected pregnant and non-pregnant mice on a protein level and could independently confirm significantly reduced IFN-α, IFN-γ, IL-6, and TNF-α levels in lungs of pregnant infected dams on day 3 p.i. compared to non-pregnant infected mice (Figures 2B–2E). Thus, key cytokines crucial in suppressing viral spread (Durbin et al., 2000Durbin J.E. Fernandez-Sesma A. Lee C.K. Rao T.D. Frey A.B. Moran T.M. Vukmanovic S. García-Sastre A. Levy D.E. Type I IFN modulates innate and specific antiviral immunity.J. Immunol. 2000; 164: 4220-4228Crossref PubMed Scopus (232) Google Scholar) are downregulated during pregnancy in 2009 pH1N1 infected dams. Next, we assessed the frequencies of leukocyte subsets in the lungs of infected mice at different days p.i. The overall frequencies of granulocytes, macrophages (MΦ), and DCs were generally higher in the lungs of pregnant infected mice, compared to infected, non-pregnant mice (data not shown). However, we observed a significantly reduced frequency of alveolar MΦ (alvMΦ) and distinct DC subsets expressing the costimulatory markers CD40, CD86, and CD80 in pH1N1 infected pregnant compared to infected non-pregnant mice (Figures 2F and 2G). The maternal immune adaptation to pregnancy is significantly modulated by the pregnancy hormone progesterone (Arck and Hecher, 2013Arck P.C. Hecher K. Fetomaternal immune cross-talk and its consequences for maternal and offspring’s health.Nat. Med. 2013; 19: 548-556Crossref PubMed Scopus (406) Google Scholar). Hence, in order to assess whether the observed innate cell defects are associated with pregnancy hormones and have functional consequences for influenza survival and morbidity during pregnancy, we utilized mice that lack the progesterone receptor (Pgr) specifically on CD11c+ DCs (PgrfloxCD11ccre mice). We observed a—albeit marginal—higher survival during pH1N1 infection in allogenic pregnant PgrfloxCD11ccre mice, compared to infected, pregnant mice of the control strain (Figures 2H and 2I). Also, pregnant, infected PgrfloxCD11ccre mice revealed a slightly higher expression of the costimulatory marker CD80 and CD86 on alvMΦ and resDC in the lung (Figures 2J and 2K), similar to the expression on CD11c cells in non-pregnant infected wild-type mice (Figures 2F and 2G). The expression of CD40 was unaffected between mutant and control mice. The observation in pregnant, infected PgrfloxCD11ccre mice suggests that the high levels of progesterone during pregnancy account—at least in part—for the impaired innate immune response seen in infected, pregnant mice. We could independently show that injection of infected, non-pregnant mice with a progesterone derivative (dydrogesterone) in order to mirror pregnancy levels showed an increased mortality compared to sham-treated, infected mice (Figures 2L and 2M), suggesting that progesterone is involved in dampening the host’s response against pH1N1. We also tested whether humoral immunity is affected in pregnant infected mice and observed a diminished humoral/B cell response during pregnancy, mirrored by a lower frequency of total splenic B lymphocytes, lower virus-specific hemagglutination inhibition (HI) titers, and a reduced co-expression of B cell-activation markers (Figure S2). When assessing the total number of leukocytes in the lung, we observed a reduction in infected pregnant mice at 3 and 4 d.p.i. (Figures 3A and 3B ). This reduction also affected the frequency of CD8+ T cells at 3 d.p.i. (Figures 3C and 3D). This led us to assess the expression of the chemoattractants for activated T cells, Cxcl9 and Cxcl10 (Loetscher et al., 1996Loetscher M. Gerber B. Loetscher P. Jones S.A. Piali L. Clark-Lewis I. Baggiolini M. Moser B. Chemokine receptor specific for IP10 and mig: structure, function, and expression in activated T-lymphocytes.J. Exp. Med. 1996; 184: 963-969Crossref PubMed Scopus (1059) Google Scholar, Bonecchi et al., 1998Bonecchi R. Bianchi G. Bordignon P.P. D’Ambrosio D. Lang R. Borsatti A. Sozzani S. Allavena P. Gray P.A. Mantovani A. Sinigaglia F. Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s.J. Exp. Med. 1998; 187: 129-134Crossref PubMed Scopus (1835) Google Scholar). We detected a significantly reduced expression of Cxcl10 in lungs of pregnant, infected mice (Figure 3E), confirming our observations from the transcriptome analyses (Table S1). Similarly, the expression of Cxcl9 was also reduced (Figure 3F). Based on these findings, we next tested whether the homing of CD8+ T cells into the bona fide tissue effector site for CD8+ T cells during influenza infection, the lung, is reduced (Figure 3G). We observed a significantly reduced frequency of migrated CD8+ T cells bearing the cognate Cxcl10 receptor, CXCR3, to the lung (Figures 3H and 3I). In order to test if the ligands for CXCR3, Cxcl9 and Cxcl10, are epigenetically silenced in the lung—similar to the observations made in the pregnant uterus (Nancy et al., 2012Nancy P. Tagliani E. Tay C.S. Asp P. Levy D.E. Erlebacher A. Chemokine gene silencing in decidual stromal cells limits T cell access to the maternal-fetal interface.Science. 2012; 336: 1317-1321Crossref PubMed Scopus (288) Google Scholar)—we analyzed the repressive histone H3 trimethyl lysine 27 (H3K27me3) enrichment at promoter regions of Cxcl9 and Cxcl10. No significant difference of H3K27me3 enrichment was detected between the lungs of pregnant and non-pregnant infected mice (Figure 3J), nor in CD45neg lung stroma cells (Figure S2). We next tested the local frequency of CD8+ T cells in the lung upon infection and observed significant increased frequencies of CD44+CD62Lneg CD8+ Teff cells at 6 d.p.i. (Figures 4A and 4B ) and virus-specific CD8+ T cells (identified by MHC class I dextramer staining) in lungs of pregnant mice at 6 and 8 d.p.i. (Figures 4C and 4D). We then assessed the effectiveness of the T cell response in an in vivo killing assay by adoptively transferring virus-peptide loaded CFSEhigh and control CSFElow splenocytes into infected non-pregnant and pregnant mice and respective controls (Figure 4E). We observed a significantly lower specific lysis of virus-peptide loaded CSFEhigh cells in pregnant infected animals compared to non-pregnant infected mice (Figures 4F and 4G). We also tested whether adoptive transfer of virus-specific CD8+ T cells from infected non-pregnant donors would improve the survival of pH1N1 infected pregnant animals (Figure 4H). Here, survival rates did not differ between pregnant infected dams receiving virus-specific CD8+ T cells or naive (mock) CD8+ T cells (Figure 4I). However, the recovery from influenza virus infection improved upon virus-specific CD8+ T cell transfer in the non-moribund pregnant mice (Figure 4J). The results described so far strongly support the concept that a series of events involved in the maternal immune response fail to mount immunity and clear pH1N1 influenza virus infection during pregnancy. In order to assess whether this failure might give rise to the emergence of novel virus variants since the influenza virus RNA-dependent RNA polymerase (RdRp) lacks proofreading activity (Gabriel and Fodor, 2014Gabriel G. Fodor E. Molecular determinants of pathogenicity in the polymerase complex.Curr. Top. Microbiol. Immunol. 2014; 385: 35-60PubMed Google Scholar), we sequenced the entire viral RNA genome obtained from infected lungs of pregnant and non-pregnant mice and could identify various synonymous and non-synonymous mutations in each of the eight viral RNA segments (Figure 5A, data not shown). In pregnant mice, the sequences for hemagglutinin (HA) and the non-structural (NS) gene-encoded proteins, NS1 and nuclear export protein (NEP), were affected (Figures 5B and 5C). In order to assess whether the detected high-frequency mutations in the NS and HA genes represent a one-time event or whether they might have biological relevance, we repeated this experiment and sequenced again the entire virus genome obtained from lungs of infected pregnant and non-pregnant mice. Repeatedly, we could detect the NS mutation as the most frequent mutation in pregnant compared to non-pregnant mice (Figure 5D). The HA mutation was not detected in the biological replicate experiment, particularly highlighting the importance of the NS mutation during pregnancy. Convergent evolution, namely independent evolution of similar features, is a strong parameter regarding the biological relevance particularly of the NS mutation in pregnancy. Therefore, we focused on the role of the NS mutation on protein function, since NS1 is a key viral determinant of type I IFN antagonism and pathogenicity (Ayllon and García-Sastre, 2015Ayllon J. García-Sastre A. The NS1 protein: a multitasking virulence factor.Curr. Top. Microbiol. Immunol. 2015; 386: 73-107PubMed Google Scholar). The NS1 R211K mutation resulted in increased viral polymerase activity (Figure 6A) and repressed IFN-β induction in human cells (Figures 6B and 6C). Consistently, cytokine expression was generally reduced in the lungs of mice infected with NS recombinant virus harboring the NS1 R211K mutation except for IFN-α (Figures 6D–6G). Most significantly, IL-6 levels were reduced at 6 d.p.i., compared to mice infected with the parental WT strain (Figure 6F). Combination of the NS mutation with HA Q223R further revealed reduced cytokine expression at 6 d.p.i. (Figures 6H–6K), particularly of IFN-α and IL-6 (Figures 6H and 6J). Next, we analyzed the biological function of the HA Q223R mutation and could show that it mediates increased binding to α2,3-linked sialic acids, which act as attachment sites for influenza viruses predominantly expressed in the lower respiratory tract (Table S2A). Then, we assessed whether these high-frequency mutations that occurred during pregnancy alter viral pathogenicity. Therefore, we infected mice with recombinant influenza viruses harboring either the HA or NS mutation or both combined. Strikingly, we observed an increased mortality and morbidity of these high-frequency pregnancy mediated mutant viruses even in non-pregnant mice compared to the parental pH1N1 virus, which was highest when both mutations encoded by HA and NS genes were combined (Figures 6L–6O and S3, and Table S2B). The increased pathogenicity observed upon combination of NS and HA mutations did not result in altered virus titers in the lungs of infected mice, but in extra-pulmonary organs (Figures 6P and 6Q), such as the gut, often observed in pathogenic 2009 pH1N1 infections in mice (Otte and Gabriel, 2011Otte A. Gabriel G. 2009 pandemic H1N1 influenza A virus strains display differential pathogenicity in C57BL/6J but not BALB/c mice.Virulence. 2011; 2: 563-566Crossref PubMed Scopus (23) Google Scholar, Otte et al., 2011Otte A. Sauter M. Alleva L. Baumgarte S. Klingel K. Gabriel G. Differential host determinants contribute to the pathogenesis of 2009 pandemic H1N1 and human H5N1 influenza A viruses in experimental mouse models.Am. J. Pathol. 2011; 179: 230-239Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). However, the single NS mutation was sufficient to mediate 100% lethality in pregnant mice (Figure S3). Finally, we could confirm that the NS1 R211K mutation, which evolved independently to elevated frequencies during pregnancy, was not present in influenza virus strains, which circulated before the pandemic occurrence in the human population (Table S2C).Figure 6In Vitro and In Vivo Characterization of the Viral High-Frequency Variants that Evolved during PregnancyShow full caption(A) The polymerase activity of pH1N1 was analyzed in H1299 cells co-expressing NS1 WT or NS1 R211K (each 2 μg). Activity of viral ribonucleoproteins in cells transfected with pcDNA3.1 empty vector as positive control (Ctr2) was set to 100%. As a second control, cells were transfected with vRNPs omitting the PB2 subunit (Ctr1). Data shown represent mean ± SD of three independent experiments performed in triplicate.(B) The IFN-β promoter activity in HEK293T transfected with NS1 WT or NS1 R211K (each 2 μg) and infected with A/WSN/33 virus (MOI 1). Activity of IFN-β promoter in cells transfected with reporter constructs and empty vector as positive control (Ctr2) was set to 100%. As a negative control, cells transfected with reporter constructs were mock infected (Ctr1). D" @default.
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• W2592337312 title "Pregnancy-Related Immune Adaptation Promotes the Emergence of Highly Virulent H1N1 Influenza Virus Strains in Allogenically Pregnant Mice" @default.
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• W2592337312 doi "https://doi.org/10.1016/j.chom.2017.02.020" @default.
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