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Summary: While severe coronavirus 2019 (COVID-19) is associated with immune activation at the maternal-fetal interface, responses to asymptomatic/mild severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during pregnancy remain unknown. Here, we assess immunological adaptations in blood and term decidua in response to asymptomatic/mild disease in pregnant women. We report attenuated antigen presentation and type I interferon (IFN) signaling pathways, loss of tissue-resident decidual macrophages, and upregulated cytokine/chemokine signaling in monocyte-derived decidual macrophages. Furthermore, we describe increased frequencies of activated tissue-resident T cells and decreased abundance of regulatory T cells with infection while frequencies of cytotoxic CD4/CD8 T cells are increased in the blood. In contrast to decidual macrophages, type I IFN signaling is higher in decidual T cells. Finally, infection leads to a narrowing of T cell receptor diversity in both blood and decidua. Collectively, these observations indicate that asymptomatic/mild COVID-19 during pregnancy results in remodeling of the immunological landscape of the maternal-fetal interface, with a potential for long-term adverse outcomes for the offspring.

ABSTRACT The ongoing pandemic of coronavirus (CoV) disease 2019 (COVID-19) continues to exert a significant burden on health care systems worldwide. With limited treatments available, vaccination remains an effective strategy to counter transmission of severe acute respiratory syndrome CoV 2 (SARS-CoV-2). Recent discussions concerning vaccination strategies have focused on identifying vaccine platforms, number of doses, route of administration, and time to reach peak immunity against SARS-CoV-2. Here, we generated a single-dose, fast-acting vesicular stomatitis virus (VSV)-based vaccine derived from the licensed Ebola virus (EBOV) vaccine rVSV-ZEBOV, expressing the SARS-CoV-2 spike protein and the EBOV glycoprotein (VSV-SARS2-EBOV). Rhesus macaques vaccinated intramuscularly (i.m.) with a single dose of VSV-SARS2-EBOV were protected within 10 days and did not show signs of COVID-19 pneumonia. In contrast, intranasal (i.n.) vaccination resulted in limited immunogenicity and enhanced COVID-19 pneumonia compared to results for control animals. While both i.m. and i.n. vaccination induced neutralizing antibody titers, only i.m. vaccination resulted in a significant cellular immune response. RNA sequencing data bolstered these results by revealing robust activation of the innate and adaptive immune transcriptional signatures in the lungs of i.m. vaccinated animals only. Overall, the data demonstrate that VSV-SARS2-EBOV is a potent single-dose COVID-19 vaccine candidate that offers rapid protection based on the protective efficacy observed in our study. IMPORTANCE The vesicular stomatitis virus (VSV) vaccine platform rose to fame in 2019, when a VSV-based Ebola virus (EBOV) vaccine was approved by the European Medicines Agency and the U.S. Food and Drug Administration for human use against the deadly disease. Here, we demonstrate the protective efficacy of a VSV-EBOV-based COVID-19 vaccine against challenge in nonhuman primates (NHPs). When a single dose of the VSV-SARS2-EBOV vaccine was administered intramuscularly (i.m.), the NHPs were protected from COVID-19 within 10 days. In contrast, if the vaccine was administered intranasally, there was no benefit from the vaccine and the NHPs developed pneumonia. The i.m. vaccinated NHPs quickly developed antigen-specific IgG, including neutralizing antibodies. Transcriptional analysis highlighted the development of protective innate and adaptive immune responses in the i.m. vaccination group only.

mRNA vaccines for SARS-CoV-2 have shown exceptional clinical efficacy, providing robust protection against severe disease. However, our understanding of transcriptional and repertoire changes following full vaccination remains incomplete. We used scRNA-Seq and functional assays to compare humoral and cellular responses to 2 doses of mRNA vaccine with responses observed in convalescent individuals with asymptomatic disease. Our analyses revealed enrichment of spike-specific B cells, activated CD4+ T cells, and robust antigen-specific polyfunctional CD4+ T cell responses following vaccination. On the other hand, although clonally expanded CD8+ T cells were observed following both vaccination and natural infection, CD8+ T cell responses were relatively weak and variable. In addition, TCR gene usage was variable, reflecting the diversity of repertoires and MHC polymorphism in the human population. Natural infection induced expansion of CD8+ T cell clones that occupy distinct clusters compared to those induced by vaccination and likely recognize a broader set of viral antigens of viral epitopes presented by the virus not seen in the mRNA vaccine. Our study highlights a coordinated adaptive immune response in which early CD4+ T cell responses facilitate the development of the B cell response and substantial expansion of effector CD8+ T cells, together capable of contributing to future recall responses.

Marburg virus (MARV) is a member of the filovirus family that causes hemorrhagic disease with high case fatality rates. MARV is on the priority list of the World Health Organization for countermeasure development highlighting its potential impact on global public health. We developed a vesicular stomatitis virus (VSV)-based vaccine expressing the MARV glycoprotein (VSV-MARV) and previously demonstrated uniform protection of nonhuman primates (NHPs) with a single dose. Here, we investigated the fast-acting potential of this vaccine by challenging NHPs with MARV 14, 7 or 3 days after a single dose vaccination with VSV-MARV. We found that 100% of the animals survived when vaccinated 7 or 14 days and 75% of the animal survived when vaccinated 3 days prior to lethal MARV challenge. Transcriptional analysis of whole blood samples indicated activation of B cells and antiviral defense after VSV-MARV vaccination. In the day -14 and -7 groups, limited transcriptional changes after challenge were observed with the exception of day 9 post-challenge in the day -7 group where we detected gene expression profiles indicative of a recall response. In the day -3 group, transcriptional analysis of samples from surviving NHPs revealed strong innate immune activation. In contrast, the animal that succumbed to disease in this group lacked signatures of antiviral immunity. In summary, our data demonstrate that the VSV-MARV is a fast-acting vaccine suitable for the use in emergency situations like disease outbreaks in Africa.

Summary: Pregravid obesity is associated with several adverse maternal health outcomes, such as increased risk of infection, suggesting an altered immunological state. However, the mechanisms by which obesity disrupts the pregnancy “immune clock” are still unknown. Here, we profiled circulating immune mediators, immune cell subset frequencies, and peripheral immune responses during the first and third trimesters of pregnancy in lean and obese mothers. While both Th1 and Th2 cytokines were elevated with pregnancy regardless of BMI, obese subjects had dysregulated myeloid factors in circulation at term. Pregnancy in lean subjects was associated with enhanced monocyte activation, augmented chromatin accessibility at inflammatory loci, and heightened responses to LPS. Pregravid obesity disrupted this trajectory, resulting in a lack of transcriptional, epigenetic, and metabolic changes strongly suggesting a skewing toward innate immune tolerance. These findings provide novel insight into the increased susceptibility to infections in women with obesity during pregnancy and following cesarean delivery.

Pregravid obesity has been shown to disrupt the development of the offspring’s immune system and increase susceptibility to infection. While the mechanisms underlying the impact of maternal obesity on fetal myeloid cells are emerging, the consequences for T cells remain poorly defined. In this study, we collected umbilical cord blood samples from infants born to lean mothers and mothers with obesity and profiled CD4 T cells using flow cytometry and single cell RNA sequencing at resting and following ex vivo polyclonal stimulation. We report that maternal obesity is associated with higher frequencies of memory CD4 T cells suggestive of in vivo activation. Moreover, single cell RNA sequencing revealed expansion of an activated subset of memory T cells with maternal obesity. However, ex vivo stimulation of purified CD4 T cells resulted in poor cytokine responses, suggesting functional defects. These phenotypic and functional aberrations correlated with methylation and chromatin accessibility changes in loci associated with lymphocyte activation and T cell receptor signaling, suggesting a possible link between maternal obesogenic environment and fetal immune reprogramming. These observations offer a potential explanation for the increased susceptibility to microbial infection in babies born to mothers with obesity.

Toxoplasma gondiiT. gondii

ABSTRACT Toxoplasma gondii can infect and replicate in vascular endothelial cells prior to entering host tissues. However, little is known about the molecular interactions at the parasite-endothelial cell interface. We demonstrate that T. gondii infection of primary human umbilical vein endothelial cells (HUVEC) altered cell morphology and dysregulated barrier function, increasing permeability to low-molecular-weight polymers. T. gondii disrupted vascular endothelial cadherin (VE-cadherin) and β-catenin localization to the cell periphery and reduced VE-cadherin protein expression. Notably, T. gondii infection led to reorganization of the host cytoskeleton by reducing filamentous actin (F-actin) stress fiber abundance under static and microfluidic shear stress conditions and by reducing planar cell polarity. RNA sequencing (RNA-Seq) comparing genome-wide transcriptional profiles of infected to uninfected endothelial cells revealed changes in gene expression associated with cell-cell adhesion, extracellular matrix reorganization, and cytokine-mediated signaling. In particular, genes downstream of Hippo signaling and the biomechanical sensor and transcriptional coactivator Yes-associated protein (YAP) were downregulated in infected endothelial cells. Interestingly, T. gondii infection activated Hippo signaling by increasing phosphorylation of LATS1, leading to cytoplasmic retention of YAP, and reducing YAP target gene expression. These findings suggest that T. gondii infection triggers Hippo signaling and YAP nuclear export, leading to an altered transcriptional profile of infected endothelial cells. IMPORTANCE Toxoplasma gondii is a foodborne parasite that infects virtually all warm-blooded animals and can cause severe disease in individuals with compromised or weakened immune systems. During dissemination in its infected hosts, T. gondii breaches endothelial barriers to enter tissues and establish the chronic infections underlying the most severe manifestations of toxoplasmosis. The research presented here examines how T. gondii infection of primary human endothelial cells induces changes in cell morphology, barrier function, gene expression, and mechanotransduction signaling under static conditions and under the physiological conditions of shear stress found in the bloodstream. Understanding the molecular interactions occurring at the interface between endothelial cells and T. gondii may provide insights into processes linked to parasite dissemination and pathogenesis.

Summary: Zaire ebolavirus (EBOV) VP35 protein is a suppressor of type I interferon (IFN) production, an inhibitor of dendritic cell maturation, and a putative virulence determinant. Here, a recombinant EBOV encoding a mutant VP35 virus (VP35m) is demonstrated to activate RIG-I-like receptor signaling and innate antiviral pathways. When inoculated into cynomolgus macaques, VP35m exhibits dramatic attenuation as compared to wild-type EBOV (wtEBOV), with 20 or 300 times the standard 100% lethal challenge dose not causing EBOV disease (EVD). Further, VP35m infection, despite limited replication in vivo, activates antigen presentation and innate immunity pathways and elicits increased frequencies of proliferating memory T cells and B cells and production of anti-EBOV antibodies. Upon wtEBOV challenge, VP35m-immunized animals survive, exhibiting host responses consistent with an orderly immune response and the absence of excessive inflammation. These data demonstrate that VP35 is a critical EBOV immune evasion factor and provide insights into immune mechanisms of EBOV control. : Woolsey et al. demonstrate that immune evasion functions of the Ebola virus VP35 protein are critical for virulence in non-human primates. A VP35 mutant Ebola virus does not cause lethal disease and instead elicits adaptive immune responses that can protect animals from wild-type Ebola virus challenge. Keywords: Ebola, filovirus, primate, VP35, RIG-I, RLR signaling, interferon, innate immunity, pathogenesis

ABSTRACT Ebola virus (EBOV) is a single-stranded RNA virus that causes Ebola virus disease (EVD), characterized by excessive inflammation, lymphocyte apoptosis, hemorrhage, and coagulation defects leading to multiorgan failure and shock. Recombinant vesicular stomatitis virus expressing the EBOV glycoprotein (VSV-EBOV), which is highly efficacious against lethal challenge in nonhuman primates, is the only vaccine that successfully completed a phase III clinical trial. Additional studies showed VSV-EBOV provides complete and partial protection to macaques immunized 7 and 3 days before EBOV challenge, respectively. However, the mechanisms by which this live-attenuated vaccine elicits rapid protection are only partially understood. To address this, we carried out a longitudinal transcriptome analysis of host responses in whole-blood samples collected from cynomolgus macaques vaccinated with VSV-EBOV 28, 21, 14, 7, and 3 days before EBOV challenge. Our findings indicate the transcriptional response to the vaccine peaks 7 days following vaccination and contains signatures of both innate antiviral immunity as well as B-cell activation. EBOV challenge 1 week after vaccination resulted in large gene expression changes suggestive of a recall adaptive immune response 14 days postchallenge. Lastly, the timing and magnitude of innate immunity and interferon-stimulated gene expression correlated with viral burden and disease outcome in animals vaccinated 3 days before challenge. IMPORTANCE Ebola virus (EBOV) is the causative agent of Ebola virus disease (EVD), a deadly disease and major public health threat worldwide. A safe and highly efficacious vesicular stomatitis virus-based vaccine against EBOV is the only platform that has successfully completed phase III clinical trials and has been used in recent and ongoing outbreaks. Earlier studies showed that antibodies are the main mode of protection when this vaccine is administered 28 days before EBOV challenge. Recently, we showed this vaccine can provide protection when administered as early as 3 days before challenge and before antibodies are detected. This study seeks to identify the mechanisms of rapid protection, which in turn will pave the way for improved vaccines and therapeutics. Additionally, this study provides insight into host gene expression signatures that could provide early biomarkers to identify infected individuals who are at highest risk of poor outcomes.

The introduction of highly active antiretroviral therapy (HAART) resulted in a significant increase in life expectancy for HIV patients. Indeed, in 2015, 45% of the HIV+ individuals in the United States were ≥55 years of age. Despite improvements in diagnosis and treatment of HIV infection, geriatric HIV+ patients suffer from higher incidence of comorbidities compared to age-matched HIV- individuals. Both chronic inflammation and dysbiosis of the gut microbiome are believed to be major contributors to this phenomenon, however carefully controlled studies investigating the impact of long-term (>10 years) controlled HIV (LTC-HIV) infection are lacking. To address this question, we profiled circulating immune cells, immune mediators, and the gut microbiome from elderly (≥55 years old) LTC-HIV+ and HIV- gay men living in the Palm Springs area. LTC-HIV+ individuals had lower frequency of circulating monocytes and CD4+ T-cells, and increased frequency CD8+ T-cells. Moreover, levels of systemic INFγ and several growth factors were increased while levels of IL-2 and several chemokines were reduced. Upon stimulation, immune cells from LTC-HIV+ individuals produced higher levels of pro-inflammatory cytokines. Last but not least, the gut microbiome of LTC-HIV+ individuals was enriched in bacterial taxa typically found in the oral cavity suggestive of loss of compartmentalization, while levels of beneficial butyrate producing taxa were reduced. Additionally, prevalence of Prevotella negatively correlated with CD4+ T-cells numbers in LTC-HIV+ individuals. These results indicate that despite long-term adherence and undetectable viral loads, LTC-HIV infection results in significant shifts in immune cell frequencies and gut microbial communities.

Marburg virus (MARV) is the causative agent of hemorrhagic fever outbreaks with high case fatality rates. Closely related to Ebola virus, MARV is a filamentous virus with a negative-sense, single-stranded RNA genome. Although extensive studies on filovirus countermeasures have been conducted, there are no licensed treatments against MARV infections. An experimental vaccine based on the recombinant vesicular stomatitis virus (VSV) expressing the MARV-Musoke glycoprotein demonstrated complete protection when a single dose was administered 28 days and up to 14 months prior to MARV challenge. Here, we analyzed the protective efficacy of an updated vaccine expressing the MARV-Angola glycoprotein (VSV-MARV). A single dose of VSV-MARV given 5 weeks before challenge provided uniform protection with no detectable viremia. The vaccine induced B and T cell proliferation and, importantly, antigen-specific IgG production. Transcriptomic signatures confirm these findings and suggest innate immunity engendered by VSV-MARV may direct the development of protective humoral immunity.

Existing models of Ebola virus disease (EVD) suggest antigen-presenting cells are initial targets of Zaire ebolavirus (ZEBOV). In vitro studies have shown that ZEBOV infection of monocytes and macrophages results in the production of inflammatory mediators, which may cause lymphocyte apoptosis. However, these findings have not been corroborated by in vivo studies. In this study, we report the first longitudinal analysis of transcriptional changes in purified monocytes, T-cells, and B-cells isolated from cynomolgus macaques following infection with ZEBOV-Makona. Our data reveal monocytes as one of the major immune cell subsets that supports ZEBOV replication in vivo. In addition, we report a marked increase in the transcription of genes involved in inflammation, coagulation, and vascular disease within monocytes, suggesting that monocytes contribute to EVD manifestations. Further, genes important for antigen presentation and regulation of immunity were downregulated, potentially subverting development of adaptive immunity. In contrast, lymphocytes, which do not support ZEBOV replication, showed transcriptional changes limited to a small number of interferon-stimulated genes (ISGs) and a failure to upregulate genes associated with an antiviral effector immune response. Collectively, these data suggest that ZEBOV-infected monocytes play a significant role in ZEBOV-Makona pathogenesis and strategies to suppress virus replication or modify innate responses to infection in these cells should be a priority for therapeutic intervention.