Pregnancy Downregulates Plasmablast Metabolic Gene Expression Following Influenza without Altering Long-Term Antibody Function

The normal response to influenza A infection is to remain asymptomatic; indeed, a study of volunteers who tested positive for antibodies against 2009 H1N1 revealed that a majority did not experience symptoms. However, studies of influenza pandemics since the 20th century, and to a lesser degree seasonal flu, suggest that pregnancy increases influenza-associated morbidity and mortality. The confounding effect of pregnancy has been well established to adversely alter the clinical course of influenza, with short term complications to mother and baby apparent, and long-term consequences to the fetus hypothesized. However, how the condition of pregnancy impacts long-term maternal anti-influenza immunity remains contentious; while vaccination studies suggest humoral responses sufficient for protection, published research on adaptive immune responses against influenza formed during pregnancy is limited. This study expands upon published work from our lab and others by comprehensively examining compartmental and systemic changes following pandemic A/California/07/2009 influenza infection. Previously, we linked seasonal influenza infection to clinical observations of adverse outcomes in pregnancy, enhanced lung and placental histopathology, and reduced control of viral replication in lungs of infected pregnant mothers. Here, we observe that the results of a lower infectious dose of pandemic influenza are similar to that of seasonal influenza. Importantly, we expand upon work which suggests infection during pregnancy may also affect humoral immunity. Our observations of reduced early antibody hemagglutinin inhibition and virus neutralization which resolved within 8 weeks of delivery demonstrate that influenza infection impacts antibody maturation mechanisms without alterations to B cell frequency or antibody secretion. This hypothesis is further supported by plasmablast transcriptional data, which demonstrates downregulated B cell metabolism and post-translational modification systems only among pregnant animals. These findings corroborate a link between adverse pregnancy outcomes and severe pathology observed during influenza infection, and propose resolving humoral deficiencies following influenza infection confounded by pregnancy. Additional studies are required to specify the involvement of plasmablast metabolism with early humoral immunity abnormalities to best guide vaccination strategies and improve our understanding of the immunological consequences of pregnancy. On day 12 of pregnancy (determined by 20% weight gain and/or presence of a copulation plug), pregnant and non-pregnant BALB/c mice were lightly anesthetized with isoflurane and infected intranasally with 30 µl 0.5xLD50 of mouse adapted A/California/07/2009, approximately 30 plaque forming units (p.f.u.). 10,000 plasmablasts (CD138+B220int) were sorted from the spleens of pregnant and non-pregnant influenza virus infected mice at 10 days post infection. Single-end RNA sequencing was performed on samples in duplicate to increase sequencing depth (101 base pair reads). Sequencing was checked for quality using FastQC (Barbaham Institute, United Kingdom). Processing of single end reads was performed within the R programming language. The Rsubread package align function (Liao et al., 2019) was used to align single end reads to the Mus musculus genome (GRCm38.93). BAM files of duplicate samples were merged prior to the counting of reads for each gene using the featureCounts function of the Rsubread package. Differential gene expression analysis was performed in R using the DESeq2 package (Love et al., 2014). Gene set enrichment analysis (Subramanian et al., 2005) was performed as previously described in pre-ranked mode with the clusterProfiler package (Yu et al., 2012). RNA sequencing was performed by the Yerkes National Primate Research Center, Non-human Primate Genomics Core.