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There remains a general misconception that the immune status of the fetus and neonate is immature or insufficient. However, emerging research in immune ontogeny prompts reconsideration of this orthodoxy, reframing this period instead as one of unique opportunity. Vaccine responses (qualitative and quantitative) vary between individuals, and across demographic cohorts. Elements of baseline immune status and function predict vaccine response – some of these factors are well described, others remain a subject of ongoing research, especially with the rapidly expanding field of ‘omics’ research, enabled by development of highly granular immune profiling techniques and increasing computational capacity. Age is one of the strongest predictive factors associated with variability in the response to vaccination; and predictable variation in response to vaccination is a key to identify the crucial underlying mechanisms. Specifically, circulating maternal antibody in the young infant can modulate immune response to vaccination, acting as an ‘undercover adjuvant’ that, counter to current dogma, may offer a pathway to longer lasting, higher quality immune response to vaccination. Exciting avenues for novel research in this area have the potential to dramatically alter how we protect the world's most vulnerable population – the very young., SCOPUS: ar.j, info:eu-repo/semantics/published

The coronavirus disease 2019 (COVID-19) pandemic continues to spread globally, highlighting the urgent need for safe and effective vaccines that could be rapidly mobilized to immunize large populations. We report the preclinical development of a self-amplifying mRNA (SAM) vaccine encoding a prefusion stabilized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein and demonstrate strong cellular and humoral immune responses at low doses in mice and rhesus macaques. The homologous prime-boost vaccination regimen of SAM at 3, 10 and 30 μg induced potent neutralizing antibody (nAb) titers in rhesus macaques following two SAM vaccinations at all dose levels, with the 10 μg dose generating geometric mean titers (GMT) 48-fold greater than the GMT of a panel of SARS-CoV-2 convalescent human sera. Spike-specific T cell responses were observed with all tested vaccine regimens. SAM vaccination provided protective efficacy against SARS-CoV-2 challenge as both a homologous prime-boost and as a single boost following ChAd prime, demonstrating reduction of viral replication in both the upper and lower airways. The SAM vaccine is currently being evaluated in clinical trials as both a homologous prime-boost regimen at low doses and as a boost following heterologous prime.

Probiotics are increasingly recognized as capable of positively modulating several aspects of human health. There are numerous attributes that make an ideal probiotic. Lactiplantibacillus plantarum (Lp) exhibits an ecological and metabolic flexibility that allows it to thrive in a variety of environments. The present review will highlight the genetic and functional characteristics of Lp that make it an ideal probiotic and summarizes the current knowledge about its potential application as a prophylactic or therapeutic intervention.

pepVIII, an anticancer vaccine targeting EGFRvIII, has been tested in several trials for glioblastoma including ACT IV. a randomized Phase III trial for newly diagnosed patients. While results from ACT IV were equivocal, available data suggests the elimination of EGFRvIII was not robust. Despite its essential role in antigen presentation, enhancing proteasomal processing is an unexploited strategy for improving vaccines. We examined 20 peptides in silico and experimentally, which showed that a tyrosine substitution (Y6-pepVIII) maximizes proteasome cleavage and survival in a subcutaneous tumor model. In an intracranial glioma model, Y6-pepVIII showed a 62 and 31% improvement in median survival compared to control animals and pepVIII-vaccinated mice. Y6-pepVIII vaccination altered tumor-infiltrating lymphocyte subsets and expression of PD-1 on intratumoral T cells. Combination with anti–PD-1 therapy cured 45% of the Y6-pepVIII–vaccinated mice but was ineffective for pepVIII-treated mice. LC-MS/MS analysis of proteasome-digested pepVIII and Y6-pepVIII revealed that most fragments were similar but more abundant inY6-pepVIII digests and 77% resulted from proteasome-catalyzed peptide splicing (PCPS). We identified 10 peptides that bound human and murine MHC class I. Nine were PCPS products and only one peptide was colinear with EGFRvIII, indicating that PCPS fragments may be a component of MHC class I recognition. Despite not being colinear with EGFRvIII, two of three PCPS products tested were capable of increasing survival when administered independently as vaccines. We hypothesize that the immune response to a vaccine represents the collective contribution from multiple PCPS and linear products. Our work suggests a strategy to increase proteasomal processing of a vaccine that results in an augmented immune response and enhanced survival in mice.

Based on mutant phenotypes the MONOPTEROS (MP)/Auxin Response Factor 5 (ARF5) gene acts in several developmental processes including leaf vein development. Since overlapping functions among ARF genes are common, we assessed the related ARF 3-8 and 19 genes for potential overlap in expression during vein development using in-situ hybridization. Like MP/ARF5, ARF3 was expressed in preprocambial and procambial cells. ARF7 was also expressed in procambial cells, close to and during vein differentiation. ARF19 was expressed in differentiating vessel elements. To assess if genes with vein expression have overlapping functions, double mutants were generated. While arf3, 5 and 7 mutants formed leaves normally, double mutant combinations of mp/arf5 with arf3 or arf7 resulted in a breakdown of leaf formation. Instead, novel structures not present in any of the single mutants formed. The results implicate ARF3 and ARF7 in rosette leaf formation and suggest that their functions overlap and act in parallel with MP/ARF5 in this process. The observed vascular expression patterns suggest unique functions (ARF7 and 19) and potentially overlapping functions (ARF3 and 5) in vein development. Since arf3 arf5 double mutants do not form leaves, assessment of their potential combined action in vein development will require the use of conditional mutants.

Inhibition of precursor B-cell malignancy progression by toll-like receptor ligand-induced immune responses

Key message The monoterpenoid content ofThuja plicataneedles and scales differs both quantitatively and qualitatively. Resin storage structures are associated with anatomical modifications that suggest facilitated exit of monoterpenoids.

Several retrospective studies in children with B cell precursor (BCP) acute lymphoblastic leukemia (ALL) provided clinical evidence that higher absolute lymphocyte counts (ALC) early into treatment significantly correlated with improved relapse-free and overall survival. It still remains unknown, however, whether the predictive role of higher ALCs reflects general bone marrow recovery or a more specific attribute of immune function. To investigate this question, we implemented a prospective observational cohort study in 20 children with BCP ALL on day 29 (D29) of induction chemotherapy and immunophenotyped their lymphoid (T, B and natural killer cells) and myeloid (neutrophils, monocytes, dendritic cells) compartments. In a first evaluation of a cohort treated with Children’s Oncology Group-based induction chemotherapy, the immune cell compartments were differentially depleted at D29. Neither gender, risk status, minimal residual disease, nor bone marrow recovery markers correlated with D29 ALC. In contrast, both CD3+ T cell and dendritic cell compartments, which did not correlate with age, significantly correlated with D29 ALC (p

B cell precursor acute lymphoblastic leukemia (B-ALL), the most common cancer in children, is a multi-step disease that is initiated in utero; although B-ALL has an incidence peak at 3-5 years of age, the majority of these children have pre-leukemic cells detectable at birth. Notably, however, up to 5% of newborns have detectable leukemia-initiating genetic abnormalities in their blood, but most of these infants go on to live healthy lives. Identifying the mechanisms that inhibit or drive B-ALL progression has the potential to inform new therapeutic or preventative strategies. Infection has been identified as a potential leukemia progression modifier to explain the discordance between the number of children with pre-leukemia and the number who develop B-ALL. While epidemiological studies have identified the timing of infection as a critical variable, the mechanisms underlying this association remain unknown. The early incidence peak is dominated by the ETV6-RUNX1 translocation-positive and high hyperdiploid subgroups, which together account for ~50% of pediatric B-ALL cases. We have previously reported the ability of immune stimulation with infection-associated toll-like receptor agonists to reduce pre-leukemic cell burden and delay leukemia onset in Eμ-RET mice, in which hyperdiploid B-ALL is fully penetrant. Furthermore, exposure of Eμ-RET mice to mild infections with Listeria monocytogenes (Lm), Murine herpesvirus 68 (MHV) or Murine cytomegalovirus (MCMV) induces a profound, age-dependent depletion of pre-leukemia cells, with neonatal mice, but not adults, exhibiting reduced leukemia progression after infection. Here, we extend this work by evaluating the influence of Saccharomyces cerevisiae (Sc) on leukemia development, hypothesizing that exposures to non-pathogenic commensal organisms would induce a similar protective immune activity. Using our established methodology, Eμ-RET mice were exposed to pathogenic or non-pathogenic organisms at six days of age by intraperitoneal injection (Lm, MCMV, Sc) or intranasally (MHV), and evaluated for pre-leukemic cell burden eight days later. In contrast to the response to pathogens, exposure of neonates to Sc did not result in a reduction of pre-leukemic cell numbers, even after inoculation with significantly higher numbers of yeast particles. The depletion of pre-leukemia cells induced by infection with pathogenic organisms was dependent on the presence of both γδ T cells and NK cells, with the latter population exerting direct cytotoxicity against B-ALL cells. Consistent with a failure to induce pre-leukemia depletion, Sc exposure achieved significantly less activation of γδ T cells and did not activate NK cells at all in neonatal Eμ-RET mice. Overall, our results reveal the primacy of the γδ T/NK cell response axis for the depletion of pre-leukemic cells after early-life infection and thus identify a novel immune response pathway with the potential to prevent ALL progression in children. Furthermore, these data indicate that not all age-associated exposures are capable of driving an immune response that reduces the risk of leukemia, with the dietary commensal Saccharomyces cerevisiae failing to generate a protective response in neonatal mice. This study significantly enhances our mechanistic understanding of immune-mediated modification of B-ALL progression. Disclosures No relevant conflicts of interest to declare.

A large proportion of GBM tumors express an altered, constitutively active version of the EGF receptor referred to as EGFRvIII. Along with increasing proliferation and inhibiting apoptosis, expression of EGFRvIII is also a GBM stem cell marker. Targeting tumor stem cells based on EGFRvIII expression may therefore improve survival for GBM patients. As such, several clinical trials using a peptide vaccine directed against EGFRvIII have shown promising results. We have explored improved versions of our original EGFRvIII peptide vaccine and have identified a candidate that shows a 60% improvement in murine subcutaneous tumor models. In this study, we further assessed the efficacy of this 2nd generation vaccine in a murine GL261 intracranial tumor model. Mice that received the EGFRvIII peptide vaccine survived 40% longer (median) than non-vaccinated mice. This response required both CD4+ and CD8+ T cells as the observed survival benefit was lost when either of these cell populations was depleted. The specificity of this vaccine strategy was valicated by an in vitro cytotoxicity assay which demonstrated that T cells derived from EGFRvIII peptide vaccinated mice were capable of specifically killing EGFRvIII expressing GL261 target cells in vitro, while demonstrating no cytotoxitiy against EGFRvIII negative cells. Treatment with the peptide vaccine increased the abundance of tumor infiltrating CD8+ T cells (p=0.0017) and altered the intra-tumoral CD4+ to CD8+ cell ratio (p=0.0015). Moreover, relative to control mice, tumor infiltrating CD8+ T cells in vaccinated mice show significant expression of the conventional immune checkpoint receptor PD-1, indicating that combinational therapies may potentiate the vaccine induced survival benefit in this model. We are currently assessing the utility of both adjuvant and immunomodulatory combinations in an attempt to further increase the efficacy of this second generation peptide vaccine.

Intravenous Ig is used to treat autoimmune or autoinflammatory disorders, but the mechanism by which it exerts its immunosuppressive activity is not understood completely. To examine the impact of intravenous Ig on macrophages, we compared cytokine production by LPS-activated macrophages in the presence and absence of intravenous Ig. Intravenous Ig treatment induced robust production of IL-10 in response to LPS, relative to LPS stimulation alone, and reduced production of proinflammatory cytokines. This anti-inflammatory, intravenous Ig-induced activation was sustained for 24 h but could only be induced if intravenous Ig were provided within 1 h of LPS stimulation. Intravenous Ig activation led to enhanced and prolonged activation of MAPKs, Erk1/2, p38, and Erk5, and inhibition of each reduced intravenous Ig-induced IL-10 production and suppression of IL-12/23p40. IL-10 production occurred rapidly in response to intravenous Ig + LPS and was sufficient to reduce proinflammatory IL-12/23p40 production in response to LPS. IL-10 induction and reduced IL-12/23p40 production were transcriptionally regulated. IL-10 played a direct role in reducing proinflammatory cytokine production by macrophages treated with intravenous Ig + LPS, as macrophages from mice deficient in the IL-10R β chain or in IL-10 were compromised in their ability to reduce proinflammatory cytokine production. Finally, intraperitoneal injection of intravenous Ig or intravenous Ig + LPS into mice activated macrophages to produce high levels of IL-10 during subsequent or concurrent LPS challenge, respectively. These findings identify IL-10 as a key anti-inflammatory mediator produced by intravenous Ig-treated macrophages and provide insight into a novel mechanism by which intravenous Ig may dampen down inflammatory responses in patients with autoimmune or autoinflammatory diseases.

An influence of infection on the development of pediatric acute lymphoblastic leukemia (ALL) is supported by epidemiological studies that have uncovered associations between infection history and ALL risk. However, these studies have produced discordant results, with both reduced and increased risk having been reported, and a mechanistic explanation remains elusive. Back-tracking studies have demonstrated that the development of ALL depends upon the long-term survival and evolution of an abnormal leukemia-initiating cell (LIC) population that arises in utero. However, the occurrence of detectable LICs at birth is significantly greater than the incidence of leukemia, suggesting that events during the pre-leukemic phase may influence disease progression by altering the fate of these cells. In this study, we use the B cell precursor (BCP) leukemia-prone Emu-RET transgenic mouse to investigate whether infection exposure is capable of altering disease progression via changes in LIC survival. Epidemiological studies have associated low grade, sub-clinical infections with reduced ALL risk, while more severe infections are associated with increased risk. To mimic a sub-clinical infection, we exposed Emu-RET mice to an ActA-/- attenuated strain of Listeria monocytogenes (Lm) prior to leukemia onset. Infection resulted in a significant depletion of LICs in spleen and bone marrow (p Citation Format: Mario Fidanza, Sheka Y. Aloyouni, Sehyun Cho, Alix E. Seif, Soren Gantt, Tobias R. Kollmann, Gregor SD Reid. Early-life infection delays B cell precursor leukemia onset in Eu-Ret mice via an IL-23-dependent depletion of leukemia-initiating cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A51.

Onset of pediatric acute lymphoblastic leukemia (ALL) depends on the long-term survival and evolution of a pre-leukemic cell population that first arises during fetal development. The detection of chromosomal translocations indicates that about 1% of all newborns harbor early-occurring abnormal cells; however, only 1 in 100 of these infants will progress to leukemia. This reveals that progression to leukemia is not the inevitable fate of these early-occurring abnormal cells. Infection has long been postulated to play a role in the development and progression of pediatric ALL. While epidemiologic studies have implicated exposure to infection as a protective factor, the impact of immune modulation during the pre-leukemic phase has not been reported. In this study, we use the Emu-RET transgenic mouse, which develops fetal-derived B cell precursor (BCP) leukemia, to investigate whether exposure to danger signals associated with infection influences disease progression via changes in pre-leukemic cell survival. Using splenocyte cultures established from pre-leukemic mice, we observed significant depletion of the characteristic pre-leukemic abnormal cells following incubation with a panel of ligands for infection-related pattern recognition receptors. The strongest activity was observed with ligands for Toll-like receptors (TLR) 2 (Pam3), 7/8 (R848), and 9 (CpG), with >75% reduction in pre-leukemic cell number (p To investigate the contribution of direct and indirect pathways to pre-leukemic cell depletion, we purified abnormal cells from pre-leukemic mouse spleens and cultured these cells in the presence or absence of TLR ligands. To date, significant direct cytotoxicity has been observed only with Pam3 (86% reduction, p = 0.0013), while R848 and CpG induce modest direct cytotoxicity. Importantly, however, pre-leukemic cell killing by R848 and CpG was enhanced by the presence of bone marrow from Nod-SCID/gamma common chain-deficient mice, indicating a significant contribution of indirect, lymphocyte-independent killing to the overall depletion of pre-leukemic cells (p Consistent with these in vitro results, we have previously shown that administration of CpG to leukemia-prone mice early in life reduced the size of the pre-leukemic cell population and significantly delayed onset of disease (p Our results are the first to deomnstrate that exposure to infection-related danger signals alters leukemia progression through the reduction in pre-leukemic cell viability. These findings provide mechanistic support for the protective effects of early-life infections and suggest novel strategies to eliminate the early-occurring abnormal cells that can give rise to initial disease and relapse. Citation Format: Mario Fidanza, Sumin Jo, Arnawaz Bashir, Stephan A. Grupp, Alix E. Seif, Gregor S. Reid. Toll-like receptor ligands delay acute lymphoblastic leukemia onset via depletion of pre-leukemic cells. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B33.