The vaccinia-based Sementis Copenhagen Vector coronavirus disease 2019 vaccine induces broad and durable cellular and humoral immune responses.

The ongoing coronavirus disease 2019 (COVID-19) pandemic perpetuated by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has highlighted the continued need for broadly protective vaccines that elicit robust and durable protection. Here, the vaccinia virus-based, replication-defective Sementis Copenhagen Vector (SCV) was used to develop a first-generation COVID-19 vaccine encoding the spike glycoprotein (SCV-S). Vaccination of mice rapidly induced polyfunctional CD8 T cells with cytotoxic activity and robust type 1 T helper-biased, spike-specific antibodies, which are significantly increased following a second vaccination, and contained neutralizing activity against the alpha and beta variants of concern. Longitudinal studies indicated that neutralizing antibody activity was maintained up to 9 months after vaccination in both young and middle-aged mice, with durable immune memory evident even in the presence of pre-existing vector immunity. Therefore, SCV-S vaccination has a positive immunogenicity profile, with potential to expand protection generated by current vaccines in a heterologous boost format and presents a solid basis for second-generation SCV-based COVID-19 vaccine candidates incorporating additional SARS-CoV-2 immunogens.

Macrophages exert homeostatic actions in pregnancy to protect against preterm birth and fetal inflammatory injury.

Macrophages are commonly thought to contribute to the pathophysiology of preterm labor by amplifying inflammation - but a protective role has not previously been considered to our knowledge. We hypothesized that given their antiinflammatory capability in early pregnancy, macrophages exert essential roles in maintenance of late gestation and that insufficient macrophages may predispose individuals to spontaneous preterm labor and adverse neonatal outcomes. Here, we showed that women with spontaneous preterm birth had reduced CD209+CD206+ expression in alternatively activated CD45+CD14+ICAM3- macrophages and increased TNF expression in proinflammatory CD45+CD14+CD80+HLA-DR+ macrophages in the uterine decidua at the materno-fetal interface. In Cd11bDTR/DTR mice, depletion of maternal CD11b+ myeloid cells caused preterm birth, neonatal death, and postnatal growth impairment, accompanied by uterine cytokine and leukocyte changes indicative of a proinflammatory response, while adoptive transfer of WT macrophages prevented preterm birth and partially rescued neonatal loss. In a model of intra-amniotic inflammation-induced preterm birth, macrophages polarized in vitro to an M2 phenotype showed superior capacity over nonpolarized macrophages to reduce uterine and fetal inflammation, prevent preterm birth, and improve neonatal survival. We conclude that macrophages exert a critical homeostatic regulatory role in late gestation and are implicated as a determinant of susceptibility to spontaneous preterm birth and fetal inflammatory injury.

Regulatory B Cells: Dark Horse in Pregnancy Immunotherapy?

There are many unanswered questions surrounding the function of immune cells and how they interact with the reproductive system to support successful pregnancy or contribute to pregnancy pathologies. While the role of immune cells such as uterine natural killer and dendritic cells, and more recently regulatory T cells has been established, the role of another major immune cell population, the B cell, and particularly the regulatory B cells, is relatively poorly understood. This review outlines what is known about B-cell subsets in the context of pregnancy, what constitutes a regulatory B cell and what role they may play, particularly during early pregnancy. Lastly, we discuss why immunotherapies for the treatment of pregnancy disorders is not widely progressed clinically and speculate on the potential of functional regulatory B cells as the basis of novel immunotherapeutic approaches for the treatment of immune-based pregnancy pathologies.

The vaccinia virus based Sementis Copenhagen Vector vaccine against Zika and chikungunya is immunogenic in non-human primates.

The Sementis Copenhagen Vector (SCV) is a new vaccinia virus-derived, multiplication-defective, vaccine technology assessed herein in non-human primates. Indian rhesus macaques (Macaca mulatta) were vaccinated with a multi-pathogen recombinant SCV vaccine encoding the structural polyproteins of both Zika virus (ZIKV) and chikungunya virus (CHIKV). After one vaccination, neutralising antibody responses to ZIKV and four strains of CHIKV, representative of distinct viral genotypes, were generated. A second vaccination resulted in significant boosting of neutralising antibody responses to ZIKV and CHIKV. Following challenge with ZIKV, SCV-ZIKA/CHIK-vaccinated animals showed significant reductions in viremias compared with animals that had received a control SCV vaccine. Two SCV vaccinations also generated neutralising and IgG ELISA antibody responses to vaccinia virus. These results demonstrate effective induction of immunity in non-human primates by a recombinant SCV vaccine and illustrates the utility of SCV as a multi-disease vaccine platform capable of delivering multiple large immunogens.

Polyinosinic: Polycytidylic Acid and Murine Cytomegalovirus Modulate Expression of Murine IL-10 and IL-21 in White Adipose Tissue.

White adipose tissue (WAT) produces interleukin-10 and other immune suppressors in response to pathogen-associated molecular patterns (PAMPs). It also homes a subset of B-cells specialized in the production of IL-10, referred to as regulatory B-cells. We investigated whether viral stimuli, polyinosinic: polycytidylic acid (poly(I:C)) or whole replicative murine cytomegalovirus (MCMV), could stimulate the expression of IL-10 in murine WAT using in vivo and ex vivo approaches. Our results showed that in vivo responses to systemic administration of poly(I:C) resulted in high levels of endogenously-produced IL-10 and IL-21 in WAT. In ex vivo WAT explants, a subset of B-cells increased their endogenous IL-10 expression in response to poly(I:C). Finally, MCMV replication in WAT explants resulted in decreased IL-10 levels, opposite to the effect seen with poly(I:C). Moreover, downregulation of IL-10 correlated with relatively lower number of Bregs. To our knowledge, this is the first report of IL-10 expression by WAT and WAT-associated B-cells in response to viral stimuli.

Maternal host responses to poly(I:C) during pregnancy leads to both dysfunctional immune profiles and altered behaviour in the offspring.

PROBLEM: Autism spectrum disorder (ASD)-like phenotypes in murine models are linked to elevated pro-inflammatory cytokine profiles caused by maternal immune activation (MIA), but whether MIA alters the immune response in the offspring remains unclear. METHOD OF STUDY: Polyinosinic:polycytidylic acid (poly:[IC]) was used to induce MIA in immunocompetent and control TLR3-deficient pregnant mice, and cytokine levels were measured in maternal and foetal organs. Furthermore, cytokines and behaviour responses were tested after challenge with lipopolysaccharide in 7-day-old and adult mice. RESULTS: MIA induced on E12 resulted in changes in the cytokine expression profile in maternal and foetal organs and correlated with TNFα and IL-18 dysregulation in immune organs and brains from neonatal mice born to MIA-induced dams. Such changes further correlated with altered behavioural responses in adulthood. CONCLUSION: MIA induced by pathogens during pregnancy can interfere with the development of the foetal immune and nervous systems leading to dysfunctional immune responses and behaviour in offspring.

Toll-Like Receptor-4 Antagonist (+)-Naltrexone Protects Against Carbamyl-Platelet Activating Factor (cPAF)-Induced Preterm Labor in Mice.

Spontaneous preterm labor is frequently caused by an inflammatory response in the gestational tissues elicited by either infectious or sterile agents. In sterile preterm labor, the key regulators of inflammation are not identified, but platelet-activating factor (PAF) is implicated as a potential rate-limiting effector agent. Since Toll-like receptor (TLR)-4 can amplify PAF signaling, we evaluated whether TLR4 contributes to inflammation and fetal loss in a mouse model of PAF-induced sterile preterm labor, and whether a small-molecule TLR4 inhibitor, (+)-naltrexone, can mitigate adverse PAF-induced effects. The administration of carbamyl (c)-PAF caused preterm labor and fetal loss in wild-type mice but not in TLR4-deficient mice. Treatment with (+)-naltrexone prevented preterm delivery and alleviated fetal demise in utero elicited after cPAF administered by i.p. or intrauterine routes. Pups born after cPAF and (+)-naltrexone treatment exhibited comparable rates of postnatal survival and growth to carrier-treated controls. (+)-Naltrexone suppressed the cPAF-induced expression of inflammatory cytokine genes Il1b, Il6, and Il10 in the decidua; Il6, Il12b, and Il10 in the myometrium; and Il1b and Il6 in the placenta. These data demonstrate that the TLR4 antagonist (+)-naltrexone inhibits the inflammatory cascade induced by cPAF, preventing preterm birth and perinatal death. The inhibition of TLR4 signaling warrants further investigation as a candidate strategy for fetal protection and delay of preterm birth elicited by sterile stimuli.

High mobility group box protein 1 neutralization therapy in ovine bacteremia: Lessons learned from an ovine septic shock model incorporating intensive care support.

Sepsis is a highly complex and often fatal syndrome which varies widely in its clinical manifestations, and therapies that target the underlying uncontrolled immune status in sepsis are needed. The failure of preclinical approaches to provide significant sepsis survival benefit in the clinic is often attributed to inappropriate animal disease models. It has been demonstrated that high mobility group box protein 1 (HMGB1) blockade can reduce inflammation, mortality and morbidity in experimental sepsis without promoting immunosuppression. Within this study, we explored the use of ovine anti-HMGB1 antibodies in a model of ovine septic shock incorporating intensive care supports (OSSICS). Results: Septic sheep exhibited elevated levels of HMGB1 within 12 h after the induction of sepsis. In this study, sepsis was induced in six anaesthetized adult Border Leicester × Merino ewes via intravenous instillation of E. coli and sheep monitored according to intensive care unit standard protocols for 26 h, with the requirement for noradrenaline as the primary endpoint. Septic sheep exhibited a hyperdynamic circulation, renal dysfunction, deranged coagulation profile and severe metabolic acidosis. Sheep were assigned a severity of illness score, which increased over time. While a therapeutic effect of intravenous anti-HMGB1 antibody could not be observed in this model due to limited animal numbers, a reduced bacterial dose induced a septic syndrome of much lower severity. With modifications including a reduced bacterial dose, a longer timeframe and broad spectrum antibiotics, the OSSICS model may become a robust tool for preclinical assessment of sepsis therapeutics.

Trophoblasts promote induction of a regulatory phenotype in B cells that can protect against detrimental T cell-mediated inflammation.

PROBLEM: A successful outcome to pregnancy is critically dependent on the initiation of maternal immune tolerance before embryo implantation. Cells of embryonic origin that come in contact with the uterine microenvironment can exert influence over the phenotype and function of immune cells to facilitate robust implantation; however, what influence they may have on B cells remains unknown. In this study, we investigate the effect of human trophoblast cells on B-cell phenotype and the subsequent effect on peri-implantation events. METHOD OF STUDY: We cultured purified human B cells with the first-trimester human trophoblast cell line Swan 71 to investigate trophoblast-B-cell interactions and utilized trophoblast spheroids in an in vitro implantation model of migration and invasion. RESULTS: Trophoblast-educated B cells or TE-B cells were found to consist of B cells in committed lineages such as plasmablasts and memory B cells, as well as increased proportions in subsets of CD24hi CD27+ regulatory B cells and CD19+ IL-10+ B cells. Conditioned media from the TE-B cells showed reduced production of pro-inflammatory cytokines that influenced the T-cell proliferation and cytokine production. Using trophoblast spheroids, we assessed the role of TE-B cells in trophoblast invasion and migration. Our results demonstrate a protective effect of TE-B-conditioned media against deleterious inflammation as evidenced by survival of the trophoblast spheroid in the presence of an immune assault and promotion of a migratory phenotype. CONCLUSION: We posit that trophoblast-mediated education of B cells leads to their acquisition of properties capable of modulating inflammation in the uterine environment during the peri-implantation period.

It takes a community to conceive: an analysis of the scope, nature and accuracy of online sources of health information for couples trying to conceive.

This study examined the nature and accuracy of information available across online platforms for couples trying to conceive. A consumer simulation-based investigation of English websites and social media (Facebook, Twitter, Instagram) was undertaken using common search terms identified in a pilot study. Claims about fertility and pregnancy health were then extracted from the results and analysed thematically. The accuracy of each claim was assessed independently by six fertility and conception experts, rated on a scale of 1 (not factual) to 4 (highly factual), with scores collated to produce a median rating. Claims with a median score < 3 were classified as inaccurate. The use of the terms 'trying to conceive' and '#TTC' were common identifiers on online platforms. Claims were extracted predominantly from websites (n = 89) rather than social media, with Twitter and Instagram comprising commercial elements and Facebook focused on community-based support. Thematic analysis revealed three major themes among the claims across all platforms: conception behaviour and monitoring, lifestyle and exposures, and medical. Fact-checking by the experts revealed that 40% of the information assessed was inaccurate, and that inaccuracies were more likely to be present in the conception behaviour and monitoring advice, the topics most amenable to modification. Since online information is a readily accessible and commonly utilized resource, there is opportunity for improved dissemination of evidence-based material to reach interested couples. Further cross-disciplinary and consumer-based research, such as a user survey, is required to understand how best to provide the 'trying to conceive' community with accurate information.

Uterine B Cells Exhibit Regulatory Properties During the Peri-Implantation Stage of Murine Pregnancy.

A successful outcome to pregnancy is dependent on the ability of the maternal uterine microenvironment to regulate inflammation processes and establish maternal tolerance. Recently, B cells have been shown to influence pregnancy outcomes as aberrations in their numbers and functions are associated with obstetric complications. In this study, we aimed to comprehensively examine the population frequency and phenotypic profile of B cells over the course of murine pregnancy. Our results demonstrated a significant expansion in B cells within the uterus during the peri-implantation period, accompanied by alterations in B cell phenotype. Functional evaluation of uterine B cells purified from pregnant mice at day 5.5 post-coitus established their regulatory capacity as evidenced by effective suppression of proliferation and activation of syngeneic CD4+ T cells. Flow cytometric analysis revealed that the uterine B cell population has an expanded pool of IL-10-producing B cells bearing upregulated expression of co-stimulatory molecules CD80 and CD86 and activation marker CD27. Our investigations herein demonstrate that during the critical stages surrounding implantation, uterine B cells are amplified and phenotypically modified to act in a regulatory manner that potentially contributes toward the establishment of maternal immunological tolerance in early pregnancy.

The Use of CRISPR/Cas9 Gene Editing to Confirm Congenic Contaminations in Host-Pathogen Interaction Studies.

Murine models of Salmonella enterica serovar Typhimurium infection are one of the commonest tools to study host-pathogen interactions during bacterial infections. Critically, the outcome of S. Typhimurium infection is impacted by the genetic background of the mouse strain used, with macrophages from C57BL/6 and BALB/c mice lacking the capacity to control intracellular bacterial replication. For this reason, the use of congenic strains, which mix the genetic backgrounds of naturally protected mouse strains with those of susceptible strains, has the capacity to significantly alter results and interpretation of S. Typhimurium infection studies. Here, we describe how macrophage knockout cell lines generated by CRISPR/Cas9 gene editing can help determine the contribution of background contaminations in the phenotypes of primary macrophages from congenic mice, on the outcome of S. Typhimurium infection studies. Our own experience illustrates how the CRISPR/Cas9 technology can be used to complement pre-existing knockout models, and shows that there is great merit in performing concurrent studies with both genetic models, to exclude unanticipated side-effects on host-pathogen interactions.

A vaccinia-based single vector construct multi-pathogen vaccine protects against both Zika and chikungunya viruses.

Zika and chikungunya viruses have caused major epidemics and are transmitted by Aedes aegypti and/or Aedes albopictus mosquitoes. The "Sementis Copenhagen Vector" (SCV) system is a recently developed vaccinia-based, multiplication-defective, vaccine vector technology that allows manufacture in modified CHO cells. Herein we describe a single-vector construct SCV vaccine that encodes the structural polyprotein cassettes of both Zika and chikungunya viruses from different loci. A single vaccination of mice induces neutralizing antibodies to both viruses in wild-type and IFNAR-/- mice and protects against (i) chikungunya virus viremia and arthritis in wild-type mice, (ii) Zika virus viremia and fetal/placental infection in female IFNAR-/- mice, and (iii) Zika virus viremia and testes infection and pathology in male IFNAR-/- mice. To our knowledge this represents the first single-vector construct, multi-pathogen vaccine encoding large polyproteins, and offers both simplified manufacturing and formulation, and reduced "shot burden" for these often co-circulating arboviruses.

Cytolytic DNA vaccine encoding lytic perforin augments the maturation of- and antigen presentation by- dendritic cells in a time-dependent manner.

The use of cost-effective vaccines capable of inducing robust CD8+ T cell immunity will contribute significantly towards the elimination of persistent viral infections and cancers worldwide. We have previously reported that a cytolytic DNA vaccine encoding an immunogen and a truncated mouse perforin (PRF) protein significantly augments anti-viral T cell (including CD8+ T cell) immunity. Thus, the current study investigated whether this vaccine enhances activation of dendritic cells (DCs) resulting in greater priming of CD8+ T cell immunity. In vitro data showed that transfection of HEK293T cells with the cytolytic DNA resulted in the release of lactate dehydrogenase, indicative of necrotic/lytic cell death. In vitro exposure of this lytic cell debris to purified DCs from naïve C57BL/6 mice resulted in maturation of DCs as determined by up-regulation of CD80/CD86. Using activation/proliferation of adoptively transferred OT-I CD8+ T cells to measure antigen presentation by DCs in vivo, it was determined that cytolytic DNA immunisation resulted in a time-dependent increase in the proliferation of OT-I CD8+ T cells compared to canonical DNA immunisation. Overall, the data suggest that the cytolytic DNA vaccine increases the activity of DCs which has important implications for the design of DNA vaccines to improve their translational prospects.

Production of a Chikungunya Vaccine Using a CHO Cell and Attenuated Viral-Based Platform Technology.

Vaccinia-based systems have been extensively explored for the development of recombinant vaccines. Herein we describe an innovative vaccinia virus (VACV)-derived vaccine platform technology termed Sementis Copenhagen Vector (SCV), which was rendered multiplication-defective by targeted deletion of the essential viral assembly gene D13L. A SCV cell substrate line was developed for SCV vaccine production by engineering CHO cells to express D13 and the VACV host-range factor CP77, because CHO cells are routinely used for manufacture of biologics. To illustrate the utility of the platform technology, a SCV vaccine against chikungunya virus (SCV-CHIK) was developed and shown to be multiplication-defective in a range of human cell lines and in immunocompromised mice. A single vaccination of mice with SCV-CHIK induced antibody responses specific for chikungunya virus (CHIKV) that were similar to those raised following vaccination with a replication-competent VACV-CHIK and able to neutralize CHIKV. Vaccination also provided protection against CHIKV challenge, preventing both viremia and arthritis. Moreover, SCV retained capacity as an effective mouse smallpox vaccine. In summary, SCV represents a new and safe vaccine platform technology that can be manufactured in modified CHO cells, with pre-clinical evaluation illustrating utility for CHIKV vaccine design and construction.

Therapeutic targeting of HMGB1 during experimental sepsis modulates the inflammatory cytokine profile to one associated with improved clinical outcomes.

Sepsis remains a significant health burden and a major clinical need exists for therapeutics to dampen the excessive and uncontrolled immune activation. Nuclear protein high mobility group box protein 1 (HMGB1) is released following cell death and is a late mediator in sepsis pathogenesis. While approaches targeting HMGB1 have demonstrated reduced mortality in pre-clinical models of sepsis, the impact of HMGB1 blockade on the complex septic inflammatory milieu and the development of subsequent immunosuppression remain enigmatic. Analysis of plasma samples obtained from septic shock patients established an association between increased HMGB1 and non-survival, higher APACHE II scores, and increased pro-inflammatory cytokine responses. Pre-clinically, administration of neutralising ovine anti-HMGB1 polyclonal antibodies improved survival in murine endotoxaemia and caecal ligation and puncture-induced sepsis models, and altered early cytokine profiles to one which corresponded to patterns observed in the surviving patient cohort. Additionally, anti-HMGB1 treated murine sepsis survivors were significantly more resistant to secondary bacterial infection and exhibited altered innate immune cell phenotypes and cytokine responses. These findings demonstrate that anti-HMGB1 antibodies alter inflammation in murine sepsis models and reduce sepsis mortality without potentiating immunosuppression.

An immunogenic phenotype in paternal antigen-specific CD8+ T cells at embryo implantation elicits later fetal loss in mice.

Central to pregnancy success is a state of T cell tolerance to paternal antigens, which is initiated at conception. The role and regulation of specific phenotypes of CD8+ T cells in mediating pregnancy tolerance is not clear. This study aimed to investigate the impact on pregnancy outcome of altering the cytokine environment during maternal CD8+ T cell priming in early pregnancy. Transgenic Act-mOVA male mice were mated to C57BL/6 (B6) females to generate fetuses expressing ovalbumin (OVA) as a model paternal antigen. OVA-reactive CD8+ OT-I T cells were activated in vitro with OVA in the presence of either transforming growth factor-ß1 (TGFB1) plus interleukin-10 (IL10), or IL2, to mimic normal or dysregulated uterine conditions, respectively, and transferred into pregnant mice on gestational day 3.5. OT-I T cells activated with TGFB1 and IL10, like naive OT-I T cells, did not alter embryo implantation or fetal viability. In contrast, OT-I T cells activated with IL2 caused extensive fetal loss manifesting in mid-gestation. IL2-activated OT-I T cells expressed less FOXP3 and higher interferon-γ (IFNG) than cells activated with TGFB1 and IL10. Fetal loss did not occur in females mated with B6 males, demonstrating the antigen specificity of fetal loss, and was not abrogated by maternal genetic C1q deficiency indicating a mechanism independent of antibody-mediated cytotoxicity. These data indicate that alternative phenotypes generated in maternal CD8+ T cells at the time of priming with paternal antigens can impact pregnancy outcome, such that inappropriate activation of CD8+ T cells before implantation is capable of causing antigen-specific fetal loss later in pregnancy.

Transient dominant host-range selection using Chinese hamster ovary cells to generate marker-free recombinant viral vectors from vaccinia virus.

Recombinant vaccinia viruses (rVACVs) are promising antigen-delivery systems for vaccine development that are also useful as research tools. Two common methods for selection during construction of rVACV clones are (i) co-insertion of drug resistance or reporter protein genes, which requires the use of additional selection drugs or detection methods, and (ii) dominant host-range selection. The latter uses VACV variants rendered replication-incompetent in host cell lines by the deletion of host-range genes. Replicative ability is restored by co-insertion of the host-range genes, providing for dominant selection of the recombinant viruses. Here, we describe a new method for the construction of rVACVs using the cowpox CP77 protein and unmodified VACV as the starting material. Our selection system will expand the range of tools available for positive selection of rVACV during vector construction, and it is substantially more high-fidelity than approaches based on selection for drug resistance.

Regulatory B Cells in Pregnancy: Lessons from Autoimmunity, Graft Tolerance, and Cancer.

The success of pregnancy is contingent on the maternal immune system recognizing and accommodating a growing semi-allogeneic fetus. Specialized subsets of lymphocytes capable of negative regulation are fundamental in this process, and include the regulatory T cells (Tregs) and potentially, regulatory B cells (Bregs). Most of our current understanding of the immune regulatory role of Bregs comes from studies in the fields of autoimmunity, transplantation tolerance, and cancer biology. Bregs control autoimmune diseases and can elicit graft tolerance by inhibiting the differentiation of effector T cells and dendritic cells (DCs), and activating Tregs. Furthermore, in cancer, Bregs are hijacked by neoplastic cells to promote tumorigenesis. Pregnancy therefore represents a condition that reconciles these fields-mechanisms must be in place to ensure maternal immunological tolerance throughout gravidity to allow the semi-allogeneic fetus to grow within. Thus, the mechanisms underlying Breg activities in autoimmune diseases, transplantation tolerance, and cancer may take place during pregnancy as well. In this review, we discuss the potential role of Bregs as guardians of pregnancy and propose an endocrine-modulated feedback loop highlighting the Breg-Treg-tolerogenic DC interface essential for the induction of maternal immune tolerance.

Evaluation of trained immunity by ß-1, 3 (d)-glucan on murine monocytes in vitro and duration of response in vivo.

The ß-1, 3 (d)-glucan (ß-glucan) present in the cell wall of Candida albicans induces epigenetic changes in human monocytes resulting in primed macrophages exhibiting increased cytokine responsiveness to reinfection. This phenomenon is referred to as trained immunity or innate immune memory. However, whether ß-glucan can reprogramme murine monocytes in vitro or induce lasting effects in vivo has yet to be elucidated. Thus, purified murine spleen-derived monocytes were primed with ß-glucan in vitro and assessed for markers of differentiation and survival. Important macrophage cell markers during monocyte-to-macrophage differentiation were downregulated and survival enhanced due to partial inhibition of apoptosis. Increased survival and not the ß-glucan training effect explained the elevated production of tumour necrosis factor-α (TNFα) and interleukin-6 (IL-6) induced by subsequent lipopolysaccharide (LPS) challenge. In vivo, 4 days after systemic administration of ß-glucan, mice were more responsive to LPS challenge as shown by the increased serum levels of TNFα, IL-6 and IL-10, an effect shown to be short lived as enhanced cytokine production was lost by day 20. Here, we have characterised murine macrophages derived from ß-glucan-primed monocytes based on their surface marker expression and for the first time provide evidence that the training effect of ß-glucan in vivo declines within a 3-week period.