H1N1 influenza virus infection results in adverse pregnancy outcomes by disrupting tissue-specific hormonal regulation.

Increased susceptibility to influenza virus infection during pregnancy has been attributed to immunological changes occurring before and during gestation in order to "tolerate" the developing fetus. These systemic changes are most often characterized by a suppression of cell-mediated immunity and elevation of humoral immune responses referred to as the Th1-Th2 shift. However, the underlying mechanisms which increase pregnant mothers' risk following influenza virus infection have not been fully elucidated. We used pregnant BALB/c mice during mid- to late gestation to determine the impact of a sub-lethal infection with A/Brisbane/59/07 H1N1 seasonal influenza virus on completion of gestation. Maternal and fetal health status was closely monitored and compared to infected non-pregnant mice. Severity of infection during pregnancy was correlated with premature rupture of amniotic membranes (PROM), fetal survival and body weight at birth, lung viral load and degree of systemic and tissue inflammation mediated by innate and adaptive immune responses. Here we report that influenza virus infection resulted in dysregulation of inflammatory responses that led to pre-term labor, impairment of fetal growth, increased fetal mortality and maternal morbidity. We observed significant compartment-specific immune responses correlated with changes in hormonal synthesis and regulation. Dysregulation of progesterone, COX-2, PGE2 and PGF2α expression in infected pregnant mice was accompanied by significant remodeling of placental architecture and upregulation of MMP-9 early after infection. Collectively these findings demonstrate the potential of a seasonal influenza virus to initiate a powerful pro-abortive mechanism with adverse outcomes in fetal health.

Zika virus-induced neuro-ocular pathology in immunocompetent mice correlates with anti-ganglioside autoantibodies.

A severe consequence of adult Zika virus (ZIKV) infection is Guillain-Barré Syndrome (GBS), where autoreactive antibodies attack peripheral and central nervous systems (CNS) resulting in neuro-ocular pathology and fatal complications. During virally induced GBS, autoimmune brain demyelination and macular degeneration correlate with low virus neutralization and elevated antibody-mediated infection among Fcγ-R bearing cells. The use of interferon-deficient mice for ZIKV studies limits elucidation of antibody-dependent enhancement (ADE) and long-term pathology (≥120 days), due to high lethality post-infection. Here we used immunocompetent BALB/c mice, which generate robust humoral immune responses, to investigate long-term impacts of ZIKV infection. A high infectious dose (1x106 FFU per mouse) of ZIKV was administered intravenously. Control animals received a single dose of anti-IFNAR blocking monoclonal antibody and succumbed to lethal neurological pathology within 13 days. Immunocompetent mice exhibited motor impairment such as arthralgia, as well as ocular inflammation resulting in retinal vascular damage, and corneal edema. This pathology persisted 100 days after infection with evidence of chronic inflammation in immune-privileged tissues, demyelination in the hippocampus and motor cortex regions of the brain, and retinal/corneal hyperplasia. Anti-inflammatory transcriptional responses were tissue-specific, likely contributing to differential pathology in these organs. Pathology in immunocompetent animals coincided with weakly neutralizing antibodies and increased ADE among ZIKV strains (PRVABC59, FLR, and MR766) and all Dengue virus (DENV) serotypes. These antibodies were autoreactive to GBS-associated gangliosides. This study highlights the importance of longevity studies in ZIKV infection and confirms the role of anti-ganglioside antibodies in ZIKV-induced neuro-ocular disease.

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

While the majority of influenza-infected individuals show no or mild symptomatology, pregnant women are at higher risk of complications and infection-associated mortality. Although enhanced lung pathology and dysregulated hormones are thought to underlie adverse pregnancy outcomes following influenza infection, how pregnancy confounds long-term maternal anti-influenza immunity remains to be elucidated. Previously, we linked seasonal influenza infection to clinical observations of adverse pregnancy outcomes, enhanced lung and placental histopathology, and reduced control of viral replication in lungs of infected pregnant mothers. Here, we expand on this work and demonstrate that lower infectious doses of the pandemic A/California/07/2009 influenza virus generated adverse gestational outcomes similar to higher doses of seasonal viruses. Mice infected during pregnancy demonstrated lower hemagglutination inhibition and neutralizing antibody titers than non-pregnant animals until 63 days post infection. These differences in humoral immunity suggest that pregnancy impacts antibody maturation mechanisms without alterations to B cell frequency or antibody secretion. This is further supported by transcriptional analysis of plasmablasts, which demonstrate downregulated B cell metabolism and post-translational modification systems only among pregnant animals. In sum, these findings corroborate a link between adverse pregnancy outcomes and severe pathology observed during pandemic influenza infection. Furthermore, our data propose that pregnancy directly confounds humoral responses following influenza infection which resolves post-partem. 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.

Cutaneous vaccination ameliorates Zika virus-induced neuro-ocular pathology via reduction of anti-ganglioside antibodies.

Zika virus (ZIKV) causes moderate to severe neuro-ocular sequelae, with symptoms ranging from conjunctivitis to Guillain-Barré Syndrome (GBS). Despite the international threat ZIKV poses, no licensed vaccine exists. As ZIKV and DENV are closely related, antibodies against one virus have demonstrated the ability to enhance the other. To examine if vaccination can confer robust, long-term protection against ZIKV, preventing neuro-ocular pathology and long-term inflammation in immune-privileged compartments, BALB/c mice received two doses of unadjuvanted inactivated whole ZIKV vaccine (ZVIP) intramuscularly (IM) or cutaneously with dissolving microneedle patches (MNP). MNP immunization induced significantly higher B and T cell responses compared to IM vaccination, resulting in increased antibody titers with greater avidity for ZPIV as well as increased numbers of IFN-γ, TNF-α, IL- and IL-4 secreting T cells. When compared to IM vaccination, antibodies generated by cutaneous vaccination demonstrated greater neutralization activity, increased cross-reactivity with Asian and African lineage ZIKV strains (PRVABC59, FLR, and MR766) and Dengue virus (DENV) serotypes, limited ADE, and lower reactivity to GBS-associated gangliosides. MNP vaccination effectively controlled viremia and inflammation, preventing neuro-ocular pathology. Conversely, IM vaccination exacerbated ocular pathology, resulting in uncontrolled, long-term inflammation. Importantly, neuro-ocular pathology correlated with anti-ganglioside antibodies implicated in demyelination and GBS. This study highlights the importance of longevity studies in ZIKV immunization, and the need of exploring alternative vaccination platforms to improve the quality of vaccine-induced immune responses.

Mesenchyme-free expansion and transplantation of adult alveolar progenitor cells: steps toward cell-based regenerative therapies.

Alveolar type-2 (AT2) cells are necessary for the lung's regenerative response to epithelial insults such as influenza. However, current methods to expand these cells rely on mesenchymal co-culture, complicating the possibility of transplantation following acute injury. Here we developed several mesenchyme-free culture conditions that promote growth of murine AT2 organoids. Transplanting dissociated AT2 organoids into influenza-infected mice demonstrated that organoids engraft and either proliferate as AT2 cells or unexpectedly adopt a basal cell-like fate associated with maladaptive regeneration. Alternatively, transplanted primary AT2 cells also robustly engraft, maintaining their AT2 lineage while replenishing the alveolar type-1 (AT1) cell population in the epithelium. Importantly, pulse oximetry revealed significant increase in blood-oxygen saturation in primary AT2 recipients, indicating that transplanted cells also confer increased pulmonary function after influenza. We further demonstrated that both acid installation and bleomycin injury models are also amenable to AT2 transplantation. These studies provide additional methods to study AT2 progenitor potential, while serving as proof-of-principle for adoptive transfer of alveolar progenitors in potential therapeutic applications.

Hormonal Regulation of Physiology, Innate Immunity and Antibody Response to H1N1 Influenza Virus Infection During Pregnancy.

In 2009, the H1N1 swine flu pandemic highlighted the vulnerability of pregnant women to influenza viral infection. Pregnant women infected with influenza A virus were at increased risk of hospitalization and severe acute respiratory distress syndrome (ARDS), which is associated with high mortality, while their newborns had an increased risk of pre-term birth or low birth weight. Pregnant women have a unique immunological profile modulated by the sex hormones required to maintain pregnancy, namely progesterone and estrogens. The role of these hormones in coordinating maternal immunotolerance in uterine tissue and cellular subsets has been well researched; however, these hormones have wide-ranging effects outside the uterus in modulating the immune response to disease. In this review, we compile research findings in the clinic and in animal models that elaborate on the unique features of H1N1 influenza A viral pathogenesis during pregnancy, the crosstalk between innate immune signaling and hormonal regulation during pregnancy, and the role of pregnancy hormones in modulating cellular responses to influenza A viral infection at mid-gestation. We highlight the ways in which lung architecture and function is stressed by pregnancy, increasing baseline inflammation prior to infection. We demonstrate that infection disrupts progesterone production and upregulates inflammatory mediators, such as cyclooxygenase-2 (COX-2) and prostaglandins, resulting in pre-term labor and spontaneous abortions. Lastly, we profile the ways in which pregnancy alters innate and adaptive cellular immune responses to H1N1 influenza viral infection, and the ways in which these protect fetal development at the expense of effective long-term immune memory. Thus, we highlight advancements in the field of reproductive immunology in response to viral infection and illustrate how that knowledge might be used to develop more effective post-infection therapies and vaccination strategies.

Stable incorporation of GM-CSF into dissolvable microneedle patch improves skin vaccination against influenza.

The widely used influenza subunit vaccine would benefit from increased protection rates in vulnerable populations. Skin immunization by microneedle (MN) patch can increase vaccine immunogenicity, as well as increase vaccination coverage due to simplified administration. To further increase immunogenicity, we used granulocyte-macrophage colony stimulating factor (GM-CSF), an immunomodulatory cytokine already approved for skin cancer therapy and cancer support treatment. GM-CSF has been shown to be upregulated in skin following MN insertion. The GM-CSF-adjuvanted vaccine induced robust and long-lived antibody responses cross-reactive to homosubtypic and heterosubtypic influenza viruses. Addition of GM-CSF resulted in increased memory B cell persistence relative to groups given influenza vaccine alone and led to rapid lung viral clearance following lethal infection with homologous virus in the mouse model. Here we demonstrate that successful incorporation of the thermolabile cytokine GM-CSF into MN resulted in improved vaccine-induced protective immunity holding promise as a novel approach to improved influenza vaccination. To our knowledge, this is the first successful incorporation of a cytokine adjuvant into dissolvable MNs, thus advancing and diversifying the rapidly developing field of MN vaccination technology.

Microneedle patch delivery of influenza vaccine during pregnancy enhances maternal immune responses promoting survival and long-lasting passive immunity to offspring.

Influenza virus causes life-threatening infections in pregnant women and their newborns. Immunization during pregnancy is the most effective means of preventing maternal and infant mortality/morbidity; however, influenza vaccination rates of pregnant women remain under 50%. Furthermore, the availability of vaccines in low-resource populations is limited. Skin immunization with microneedle patches (MN) is a novel and safe vaccination platform featuring thermostable vaccine formulations. Cold-chain independence and the potential for self-administration can expand influenza vaccination coverage in developing countries. In this study of pregnant BALB/c mice immunized with subunit H1N1 influenza vaccine, we demonstrate the advantage of skin vaccination over intramuscular delivery of a two-fold higher vaccine dose. MN vaccine induced superior humoral immune responses and conferred protective immunity against a lethal challenge dose of homologous influenza virus. Importantly, MN vaccination of mice at mid-gestation resulted in enhanced and long-lasting passive immunity of the offspring, measured by neutralizing antibody titers and survival rates after virus challenge. We conclude that skin vaccination using MN is a superior immunization approach with the potential to overcome immune tolerance observed in pregnancy, and lower vaccination costs through antigen dose-sparing, which is especially relevant in underserved countries.