Single-cell proteomics and transcriptomics capture eosinophil development and identify the role of IL-5 in their lineage transit amplification.

The activities, ontogeny, and mechanisms of lineage expansion of eosinophils are less well resolved than those of other immune cells, despite the use of biological therapies targeting the eosinophilia-promoting cytokine interleukin (IL)-5 or its receptor, IL-5Rα. We combined single-cell proteomics and transcriptomics and generated transgenic IL-5Rα reporter mice to revisit eosinophilopoiesis. We reconciled human and murine eosinophilopoiesis and provided extensive cell-surface immunophenotyping and transcriptomes at different stages along the continuum of eosinophil maturation. We used these resources to show that IL-5 promoted eosinophil-lineage expansion via transit amplification, while its deletion or neutralization did not compromise eosinophil maturation. Informed from our resources, we also showed that interferon response factor-8, considered an essential promoter of myelopoiesis, was not intrinsically required for eosinophilopoiesis. This work hence provides resources, methods, and insights for understanding eosinophil ontogeny, the effects of current precision therapeutics, and the regulation of eosinophil development and numbers in health and disease.

Parasitic worms affect virus coinfection: a mechanistic overview.

Helminths are parasitic worms that coevolve with their host, usually resulting in long-term persistence through modulating host immunity. The multifarious mechanisms altering the immune system induced by helminths have significant implications on the control of coinfecting pathogens such as viruses. Here, we explore the recent literature to highlight the main immune alterations and mechanisms that affect the control of viral coinfection. Insights from these mechanisms are valuable in the understanding of clinical observations in helminth-prevalent areas and in the design of new therapeutic and vaccination strategies to control viral diseases.

Wildebeest-Derived Malignant Catarrhal Fever: A Bovine Peripheral T Cell Lymphoma Caused by Cross-Species Transmission of Alcelaphine Gammaherpesvirus 1.

Gammaherpesviruses (γHVs) include viruses that can induce lymphoproliferative diseases and tumors. These viruses can persist in the long term in the absence of any pathological manifestation in their natural host. Alcelaphine gammaherpesvirus 1 (AlHV-1) belongs to the genus Macavirus and asymptomatically infects its natural host, the wildebeest (Connochaetes spp.). However, when transmitted to several susceptible species belonging to the order Artiodactyla, AlHV-1 is responsible for the induction of a lethal lymphoproliferative disease, named wildebeest-derived malignant catarrhal fever (WD-MCF). Understanding the pathogenic mechanisms responsible for the induction of WD-MCF is important to better control the risks of transmission and disease development in susceptible species. The aim of this review is to synthesize the current knowledge on WD-MCF with a particular focus on the mechanisms by which AlHV-1 induces the disease. We discuss the potential mechanisms of pathogenesis from viral entry into the host to the maintenance of viral genomes in infected CD8+ T lymphocytes, and we present current hypotheses to explain how AlHV-1 infection induces a peripheral T cell lymphoma-like disease.

A Vaccine Targeting Ovine Herpesvirus 2 Glycoprotein B Protects against Sheep-Associated Malignant Catarrhal Fever.

Malignant catarrhal fever (MCF) is a complex and often fatal disease of ungulates. Effective vaccines are needed to avoid MCF outbreaks and mitigate losses. This study aimed to evaluate a sheep-associated MCF (SA-MCF) vaccine candidate targeting ovine herpesvirus 2 (OvHV-2) glycoprotein B (gB). Rabbits were used as a laboratory animal model to test the safety, immunogenicity, and protective efficacy of a chimeric virus consisting of a recombinant, non-pathogenic strain of alcelaphine herpesvirus-1 encoding OvHV-2 ORF8 to express gB (AlHV-1∆ORF73/OvHV-2-ORF8). Viral-vectored immunizations were performed by using the AlHV-1∆ORF73/OvHV-2-ORF8 chimera alone or as a DNA prime (OvHV-2-ORF8)-virus boost regimen. The viral vector was inoculated by intravenous or intramuscular routes and the DNA was delivered by intradermal shots using a gene gun. The vaccine candidates were deemed safe as no clinical signs were observed following any of the immunizations. Anti-OvHV-2 gB antibodies with neutralizing activity were induced by all immunogens. At three weeks post-final immunization, all animals were challenged intranasally with a lethal dose of OvHV-2. MCF protection rates ranging from 66.7% to 71.4% were observed in vaccinated rabbits, while all mock-vaccinated animals developed the disease. The significant protective efficacy obtained with the vaccine platforms tested in this study encourages further trials in relevant livestock species, such as cattle and bison.

Development of a Specific Anti-capsid Antibody- and Magnetic Bead-Based Immunoassay to Detect Human Norovirus Particles in Stool Samples and Spiked Mussels via Flow Cytometry.

Human noroviruses impose a considerable health burden globally. Here, a flow cytometry approach designed for their detection in biological waste and food samples was developed using antibody-coated magnetic beads. Antipeptide antibodies against murine norovirus and various human norovirus genotypes were generated for capture and coated onto magnetic beads. A flow cytometry assay was then implemented to detect bead-bound human norovirus GI.3 in patient stool samples and in norovirus-spiked mussel digestive tissues. The detection limit for stool samples was 105 gc/mL, thus bettering detection limits of commercially available norovirus diagnosis quick kits of 100-fold; the detection limit in spiked mussels however was ten-fold higher than in stool samples. Further assays showed a decrease in fluorescence intensity for heat- or UV-inactivated virus particles. Overall, we demonstrate the application of a flow cytometry approach for direct detection of small non-enveloped virus particles such as noroviruses. An adaptation of the technology to routine diagnostics has the potential to contribute a rapid and sensitive tool to norovirus outbreak investigations. Further improvements to the method, notably decreasing the detection limit of the approach, may allow the analysis of naturally contaminated food and environmental samples.

Il4ra-independent vaginal eosinophil accumulation following helminth infection exacerbates epithelial ulcerative pathology of HSV-2 infection.

How helminths influence the pathogenesis of sexually transmitted viral infections is not comprehensively understood. Here, we show that an acute helminth infection (Nippostrongylus brasiliensis [Nb]) induced a type 2 immune profile in the female genital tract (FGT). This leads to heightened epithelial ulceration and pathology in subsequent herpes simplex virus (HSV)-2 infection. This was IL-5-dependent but IL-4 receptor alpha (Il4ra) independent, associated with increased FGT eosinophils, raised vaginal IL-33, and enhanced epithelial necrosis. Vaginal eosinophil accumulation was promoted by IL-33 induction following targeted vaginal epithelium damage from a papain challenge. Inhibition of IL-33 protected against Nb-exacerbated HSV-2 pathology. Eosinophil depletion reduced IL-33 release and HSV-2 ulceration in Nb-infected mice. These findings demonstrate that Nb-initiated FGT eosinophil recruitment promotes an eosinophil, IL-33, and IL-5 inflammatory circuit that enhances vaginal epithelial necrosis and pathology following HSV-2 infection. These findings identify a mechanistic framework as to how helminth infections can exacerbate viral-induced vaginal pathology.

A gammaherpesvirus licenses CD8 T cells to protect the host from pneumovirus-induced immunopathologies.

Human respiratory syncytial virus (RSV) is a pneumovirus that causes severe infections in infants worldwide. Despite intensive research, safe and effective vaccines against RSV have remained elusive. The main reason is that RSV infection of children previously immunized with formalin-inactivated-RSV vaccines has been associated with exacerbated pathology, a phenomenon called RSV vaccine-enhanced respiratory disease. In parallel, despite the high RSV prevalence, only a minor proportion of children develop severe diseases. Interestingly, variation in the immune responses against RSV or following RSV vaccination could be linked with differences of exposure to microbes during childhood. Gammaherpesviruses (γHVs), such as the Epstein-Barr virus, are persistent viruses that deeply influence the immune system of their host and could therefore affect the development of pneumovirus-induced immunopathologies for the long term. Here, we showed that a previous ɣHV infection protects against both pneumovirus vaccine-enhanced disease and pneumovirus primary infection and that CD8 T cells are essential for this protection. These observations shed a new light on the understanding of pneumovirus-induced diseases and open new perspectives for the development of vaccine strategies.

Alcelaphine herpesvirus 1 genes A7 and A8 regulate viral spread and are essential for malignant catarrhal fever.

Alcelaphine herpesvirus 1 (AlHV-1) is a gammaherpesvirus that is carried asymptomatically by wildebeest. Upon cross-species transmission to other ruminants, including domestic cattle, AlHV-1 induces malignant catarrhal fever (MCF), which is a fatal lymphoproliferative disease resulting from proliferation and uncontrolled activation of latently infected CD8+ T cells. Two laboratory strains of AlHV-1 are used commonly in research: C500, which is pathogenic, and WC11, which has been attenuated by long-term maintenance in cell culture. The published genome sequence of a WC11 seed stock from a German laboratory revealed the deletion of two major regions. The sequence of a WC11 seed stock used in our laboratory also bears these deletions and, in addition, the duplication of an internal sequence in the terminal region. The larger of the two deletions has resulted in the absence of gene A7 and a large portion of gene A8. These genes are positional orthologs of the Epstein-Barr virus genes encoding envelope glycoproteins gp42 and gp350, respectively, which are involved in viral propagation and switching of cell tropism. To investigate the degree to which the absence of A7 and A8 participates in WC11 attenuation, recombinant viruses lacking these individual functions were generated in C500. Using bovine nasal turbinate and embryonic lung cell lines, increased cell-free viral propagation and impaired syncytia formation were observed in the absence of A7, whereas cell-free viral spread was inhibited in the absence of A8. Therefore, A7 appears to be involved in cell-to-cell viral spread, and A8 in viral cell-free propagation. Finally, infection of rabbits with either mutant did not induce the signs of MCF or the expansion of infected CD8+ T cells. These results demonstrate that A7 and A8 are both essential for regulating viral spread and suggest that AlHV-1 requires both genes to efficiently spread in vivo and reach CD8+ T lymphocytes and induce MCF.

Pre-conception maternal helminth infection transfers via nursing long-lasting cellular immunity against helminths to offspring.

Maternal immune transfer is the most significant source of protection from early-life infection, but whether maternal transfer of immunity by nursing permanently alters offspring immunity is poorly understood. Here, we identify maternal immune imprinting of offspring nursed by mothers who had a pre-conception helminth infection. Nursing of pups by helminth-exposed mothers transferred protective cellular immunity to these offspring against helminth infection. Enhanced control of infection was not dependent on maternal antibody. Protection associated with systemic development of protective type 2 immunity in T helper 2 (TH2) impaired IL-4Rα-/- offspring. This maternally acquired immunity was maintained into maturity and required transfer (via nursing) to the offspring of maternally derived TH2-competent CD4 T cells. Our data therefore reveal that maternal exposure to a globally prevalent source of infection before pregnancy provides long-term nursing-acquired immune benefits to offspring mediated by maternally derived pathogen-experienced lymphocytes.

Recruitment of hepatic macrophages from monocytes is independent of IL-4Rα but is associated with ablation of resident macrophages in schistosomiasis.

Alternatively activated Mφs (AAMφ) accumulate in hepatic granulomas during schistosomiasis and have been suggested to originate in the bone marrow. What is less understood is how these Mφ responses are regulated after S. mansoni infection. Here, we investigated the role of IL-4 receptor α-chain (IL-4Rα)-signalling in the dynamics of liver Mφ responses. We observed that IL-4Rα signalling was dispensable for the recruitment of Ly6Chi monocytes and for their conversion into F4/80hi CD64hi CD11bhi Mφ. Moreover, while IL-4Rα provided an AAMφ phenotype to liver F4/80hi CD64hi CD11bhi Mφ that was associated with regulation of granuloma formation, it was dispensable for host survival. Resident F4/80hi CD64hi CD11blo Mφ did not upregulate the AAMφ signature gene Ym1. Rather, resident Mφ nearly disappeared by week 8 after infection and artificial ablation of resident Mφ in CD169DTR mice did not affect the response to S. mansoni infection. Interestingly, ablation of CD169+ cells in naive mice resulted in the accumulation of F4/80hi CD64hi CD11bhi Mφ, which was amplified when ablation occurred during schistosomiasis. Altogether, our results suggest the ablation of resident KCs after S. mansoni infection to be associated with the recruitment and accumulation of F4/80hi CD64hi CD11bhi Mφ with lyz2-dependent IL-4Rα contributing to the regulation of granuloma inflammation but being dispensable for host survival.

Maternal helminth infections and the shaping of offspring immunity.

Helminth infections leave a long-lasting immunological footprint on their hosts. Clinical studies have provided first evidence that maternal helminth infections can result in an altered immune profile in their offspring which can potentially shape how they respond to conditions throughout life. This can relate to changes in offspring induction of immune responses against other diseases. However, whether these changes result in actual changes in offspring ability to control disease is unclear. Our understanding of which immune mechanisms are altered and how they are changed is limited. In this review, we highlight what we know from human and mouse studies about this important context of helminth exposure. Moreover, we discuss how mechanisms such as antibody transfer, antigen exposure, maternal cell uptake, chimerism and epigenetics are all likely to be functional contributors to the striking changes that are seen in offspring born or nursed by helminth exposed mothers.

Helminth-induced IL-4 expands bystander memory CD8+ T cells for early control of viral infection.

Infection with parasitic helminths can imprint the immune system to modulate bystander inflammatory processes. Bystander or virtual memory CD8+ T cells (TVM) are non-conventional T cells displaying memory properties that can be generated through responsiveness to interleukin (IL)-4. However, it is not clear if helminth-induced type 2 immunity functionally affects the TVM compartment. Here, we show that helminths expand CD44hiCD62LhiCXCR3hiCD49dlo TVM cells through direct IL-4 signaling in CD8+ T cells. Importantly, helminth-mediated conditioning of TVM cells provided enhanced control of acute respiratory infection with the murid gammaherpesvirus 4 (MuHV-4). This enhanced control of MuHV-4 infection could further be explained by an increase in antigen-specific CD8+ T cell effector responses in the lung and was directly dependent on IL-4 signaling. These results demonstrate that IL-4 during helminth infection can non-specifically condition CD8+ T cells, leading to a subsequently raised antigen-specific CD8+ T cell activation that enhances control of viral infection.

Macrophage Activation and Functions during Helminth Infection: Recent Advances from the Laboratory Mouse.

Macrophages are highly plastic innate immune cells that adopt an important diversity of phenotypes in response to environmental cues. Helminth infections induce strong type 2 cell-mediated immune responses, characterized among other things by production of high levels of interleukin- (IL-) 4 and IL-13. Alternative activation of macrophages by IL-4 in vitro was described as an opposite phenotype of classically activated macrophages, but the in vivo reality is much more complex. Their exact activation state as well as the role of these cells and associated molecules in type 2 immune responses remains to be fully understood. We can take advantage of a variety of helminth models available, each of which have their own feature including life cycle, site of infection, or pathological mechanisms influencing macrophage biology. Here, we reviewed the recent advances from the laboratory mouse about macrophage origin, polarization, activation, and effector functions during parasitic helminth infection.

Seroprevalence of malignant catarrhal fever virus in captive wildebeest (Connochaetes sp.) in France.

Alcelaphine herpesvirus 1 (AlHV-1) is a gammaherpesvirus carried asymptomatically by wildebeests (Connochaetes sp.) in sub-Saharan Africa. Although asymptomatic in wildebeest, AlHV-1 infection in a number of other ruminant species causes a severe and fatal lymphoproliferative disease named wildebeest-derived malignant catarrhal fever (WD-MCF). Several endangered species of captive ruminants are highly susceptible to developing WD-MCF if infected by AlHV-1, which is a critical concern in zoos, game reserves and wildlife parks where wildebeests are also kept in captivity. Here, we investigated the seroprevalence of AlHV-1 in 52 captive wildebeests randomly sampled from five different zoos in France. We found 46% (24/52) seropositive animals and detected AlHV-1 DNA in one of them, demonstrating that AlHV-1 infection is present in captive wildebeests in France. In an interesting manner, the repartition of seropositive wildebeests was not homogenous between zoos with 100% (20/20) of seronegative animals in three parks. These results further highlight the importance of considering WD-MCF as a threat for clinically susceptible species and encourage for testing AlHV-1 infection in captive wildebeests as a management control strategy.

The Critical Role of Genome Maintenance Proteins in Immune Evasion During Gammaherpesvirus Latency.

Gammaherpesviruses are important pathogens that establish latent infection in their natural host for lifelong persistence. During latency, the viral genome persists in the nucleus of infected cells as a circular episomal element while the viral gene expression program is restricted to non-coding RNAs and a few latency proteins. Among these, the genome maintenance protein (GMP) is part of the small subset of genes expressed in latently infected cells. Despite sharing little peptidic sequence similarity, gammaherpesvirus GMPs have conserved functions playing essential roles in latent infection. Among these functions, GMPs have acquired an intriguing capacity to evade the cytotoxic T cell response through self-limitation of MHC class I-restricted antigen presentation, further ensuring virus persistence in the infected host. In this review, we provide an updated overview of the main functions of gammaherpesvirus GMPs during latency with an emphasis on their immune evasion properties.

Macavirus latency-associated protein evades immune detection through regulation of protein synthesis in cis depending upon its glycin/glutamate-rich domain.

Alcelaphine herpesvirus 1 (AlHV-1) is a γ-herpesvirus (γ-HV) belonging to the macavirus genus that persistently infects its natural host, the wildebeest, without inducing any clinical sign. However, cross-transmission to other ruminant species causes a deadly lymphoproliferative disease named malignant catarrhal fever (MCF). AlHV-1 ORF73 encodes the latency-associated nuclear antigen (LANA)-homolog protein (aLANA). Recently, aLANA has been shown to be essential for viral persistence in vivo and induction of MCF, suggesting that aLANA shares key properties of other γ-HV genome maintenance proteins. Here we have investigated the evasion of the immune response by aLANA. We found that a glycin/glutamate (GE)-rich repeat domain was sufficient to inhibit in cis the presentation of an epitope linked to aLANA. Although antigen presentation in absence of GE was dependent upon proteasomal degradation of aLANA, a lack of GE did not affect protein turnover. However, protein self-synthesis de novo was downregulated by aLANA GE, a mechanism directly associated with reduced antigen presentation in vitro. Importantly, codon-modification of aLANA GE resulted in increased antigen presentation in vitro and enhanced induction of antigen-specific CD8+ T cell responses in vivo, indicating that mRNA constraints in GE rather than peptidic sequence are responsible for cis-limitation of antigen presentation. Nonetheless, GE-mediated limitation of antigen presentation in cis of aLANA was dispensable during MCF as rabbits developed the disease after virus infection irrespective of the expression of full-length or GE-deficient aLANA. Altogether, we provide evidence that inhibition in cis of protein synthesis through GE is likely involved in long-term immune evasion of AlHV-1 latent persistence in the wildebeest natural host, but dispensable in MCF pathogenesis.

Type I interferon is required for T helper (Th) 2 induction by dendritic cells.

Type 2 inflammation is a defining feature of infection with parasitic worms (helminths), as well as being responsible for widespread suffering in allergies. However, the precise mechanisms involved in T helper (Th) 2 polarization by dendritic cells (DCs) are currently unclear. We have identified a previously unrecognized role for type I IFN (IFN-I) in enabling this process. An IFN-I signature was evident in DCs responding to the helminth Schistosoma mansoni or the allergen house dust mite (HDM). Further, IFN-I signaling was required for optimal DC phenotypic activation in response to helminth antigen (Ag), and efficient migration to, and localization with, T cells in the draining lymph node (dLN). Importantly, DCs generated from Ifnar1-/- mice were incapable of initiating Th2 responses in vivo These data demonstrate for the first time that the influence of IFN-I is not limited to antiviral or bacterial settings but also has a central role to play in DC initiation of Th2 responses.