Two biochemically distinct lipophosphoglycans from Leishmania braziliensis and Leishmania infantum trigger different innate immune responses in murine macrophages

BACKGROUND: The dominant, cell surface lipophosphoglycan (LPG) of Leishmania is a multifunctional molecule involved in the interaction with vertebrate and invertebrate hosts. Although the role of LPG on infection has been extensively studied, it is not known if LPG interspecies variations contribute to the different immunopathologies of leishmaniases. To investigate the issue of interspecies polymorphisms, two Leishmania species from the New World that express structural variations of side chains of LPG repeat units were examined. In this context, the procyclic form of L. braziliensis LPG (strain M2903), is devoid of side chains, while the L. infantum LPG (strain BH46) has up to three glucoses residues in the repeat units. METHODS: Mice peritoneal macrophages from Balb/c, C57BL/6 and knock-out (TLR2 -/-, TLR4 -/-) were primed with IFN-γ and stimulated with purified LPG from both species. Nitric oxide and cytokine production, MAPKs (ERK, p38 and JNK) and NF-kB activation were evaluated. RESULTS: Macrophages stimulated with L. braziliensis LPG, had a higher TNF-α, IL-1β, IL-6 and NO production than those stimulated with that of L. infantum. Furthermore, the LPGs from the two species resulted in differential kinetics of signaling via MAPK activation. L. infantum LPG exhibited a gradual activation profile, whereas L. braziliensis LPG showed a sharp but transient activation. L. braziliensis LPG was able to activate NF-kB. CONCLUSION: These data suggest that two biochemically distinct LPGs were able to differentially modulate macrophage functions

Cross-protection induced by highly conserved human B, CD4+, and CD8+ T-cell epitopes-based vaccine against severe infection, disease, and death caused by multiple SARS-CoV-2 variants of concern

BackgroundThe coronavirus disease 2019 (COVID-19) pandemic has created one of the largest global health crises in almost a century. Although the current rate of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections has decreased significantly, the long-term outlook of COVID-19 remains a serious cause of morbidity and mortality worldwide, with the mortality rate still substantially surpassing even that recorded for influenza viruses. The continued emergence of SARS-CoV-2 variants of concern (VOCs), including multiple heavily mutated Omicron sub-variants, has prolonged the COVID-19 pandemic and underscores the urgent need for a next-generation vaccine that will protect from multiple SARS-CoV-2 VOCs.MethodsWe designed a multi-epitope-based coronavirus vaccine that incorporated B, CD4+, and CD8+ T- cell epitopes conserved among all known SARS-CoV-2 VOCs and selectively recognized by CD8+ and CD4+ T-cells from asymptomatic COVID-19 patients irrespective of VOC infection. The safety, immunogenicity, and cross-protective immunity of this pan-variant SARS-CoV-2 vaccine were studied against six VOCs using an innovative triple transgenic h-ACE-2-HLA-A2/DR mouse model.ResultsThe pan-variant SARS-CoV-2 vaccine (i) is safe , (ii) induces high frequencies of lung-resident functional CD8+ and CD4+ TEM and TRM cells , and (iii) provides robust protection against morbidity and virus replication. COVID-19-related lung pathology and death were caused by six SARS-CoV-2 VOCs: Alpha (B.1.1.7), Beta (B.1.351), Gamma or P1 (B.1.1.28.1), Delta (lineage B.1.617.2), and Omicron (B.1.1.529).ConclusionA multi-epitope pan-variant SARS-CoV-2 vaccine bearing conserved human B- and T- cell epitopes from structural and non-structural SARS-CoV-2 antigens induced cross-protective immunity that facilitated virus clearance, and reduced morbidity, COVID-19-related lung pathology, and death caused by multiple SARS-CoV-2 VOCs

Glycoinositolphospholipids from Leishmania braziliensis and L. infantum: Modulation of Innate Immune System and Variations in Carbohydrate Structure

The essential role of the lipophosphoglycan (LPG) of Leishmania in innate immune response has been extensively reported. However, information about the role of the LPG-related glycoinositolphospholipids (GIPLs) is limited, especially with respect to the New World species of Leishmania. GIPLs are low molecular weight molecules covering the parasite surface and are similar to LPG in sharing a common lipid backbone and a glycan motif containing up to 7 sugars. Critical aspects of their structure and functions are still obscure in the interaction with the vertebrate host. In this study, we evaluated the role of those molecules in two medically important South American species Leishmania infantum and L. braziliensis, causative agents of visceral (VL) and cutaneous Leishmaniasis (CL), respectively. GIPLs derived from both species did not induce NO or TNF-α production by non-primed murine macrophages. Additionally, primed macrophages from mice (BALB/c, C57BL/6, TLR2−/− and TLR4−/−) exposed to GIPLs from both species, with exception to TNF-α, did not produce any of the cytokines analyzed (IL1-β, IL-2, IL-4, IL-5, IL-10, IL-12p40, IFN-γ) or p38 activation. GIPLs induced the production of TNF-α and NO by C57BL/6 mice, primarily via TLR4. Pre incubation of macrophages with GIPLs reduced significantly the amount of NO and IL-12 in the presence of IFN-γ or lipopolysaccharide (LPS), which was more pronounced with L. braziliensis GIPLs. This inhibition was reversed after PI-specific phospholipase C treatment. A structural analysis of the GIPLs showed that L. infantum has manose rich GIPLs, suggestive of type I and Hybrid GIPLs while L. braziliensis has galactose rich GIPLs, suggestive of Type II GIPLs. In conclusion, there are major differences in the structure and composition of GIPLs from L. braziliensis and L. infantum. Also, GIPLs are important inhibitory molecules during the interaction with macrophages

Lipofosfoglicanos (LPGs) de Leishmania braziliensis e Leishmania infantum na ativação de macrófagos murinos e vias de sinalização celular

Submitted by Nuzia Santos ([email protected]) on 2013-01-15T17:01:51Z No. of bitstreams: 1 Dissertacao_Izabela Coimbra Ibraim.pdf: 3600819 bytes, checksum: 7dc73c7a016be4f2bdca2b741e85a75e (MD5)Made available in DSpace on 2013-01-15T17:01:51Z (GMT). No. of bitstreams: 1 Dissertacao_Izabela Coimbra Ibraim.pdf: 3600819 bytes, checksum: 7dc73c7a016be4f2bdca2b741e85a75e (MD5) Previous issue date: 2012Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brasil.O LPG apresenta variações estruturais que são importantes para os diferentes estágios de desenvolvimento do parasito. Nas formas procíclicas, o LPG de L. braziliensis (cepa M2903), não apresenta cadeias laterais enquanto um a dois resíduos de β-glicose podem aparecer nas unidades repetitivas da forma metacíclica. O LPG de L. infantum (cepa BH46), apresenta até três cadeias laterais de glicoses nas unidades repetitivas da forma procíclica e ainda não foi caracterizado nas formas metacíclicas. Esses polimorfismos nas unidades repetitivas do LPG e seu papel na interação com o hospedeiro vertebrado e invertebrado já foram amplamente estudados, sobretudo, para as espécies do Velho Mundo. Entretanto, para a maioria das espécies do Novo Mundo, o papel desses polimorfismos no perfil imunopatológico da doença é ainda desconhecido. Este projeto teve como objetivo avaliar o estudo da interação entre os LPGs de duas espécies epidemiologicamente importantes no Brasil e macrófagos murinos. Estas incluem L. braziliensis e L. infantum, responsáveis pela forma cutânea e visceral, respectivamente. Neste estudo, os macrófagos peritoneais de camundongos BALB/c, C57BL/6 e C57BL/6 knock-out (TLR2 -/- e TLR4 -/-), foram primados com IFN- e estimulados com LPG de ambas as espécies. A produção de citocinas (IL-1β, IL-2; IL-4, IL-6, IL-10, IL-12p40, IFN- e TNF-α) foi determinada por citometria de fluxo e a concentração de nitrito pelo método de Griess. A ativação de ERK e p38 foi avaliada por Western blot. Os macrófagos estimulados com LPG de L. braziliensis, apresentaram uma maior produção de TNF-α, IL-1β, IL-6 e NO em comparação aos estimulados com LPG de L. infantum. Também foi observada uma cinética de ativação diferencial das MAPK entre os LPGs. Leishmania infantum apresentou uma ativação constante até 45 minutos após estimulação, enquanto L. braziliensis apresentou um único pico de ativação após 15 minutos. Estes dados sugerem que variações interespecíficas no LPG de Leishmania podem ter um papel importante nos eventos iniciais do compartimento imune inato do hospedeiro.The structural variations observed on LPG are important for the different parasite developmental stages. The procyclic form of L. braziliensis LPG (strain M2903), is devoid of side chains, while one to two β-glucose side chains are added in the repeat units of the metacyclic LPG. The L. infantum LPG has with up to 3 glucoses residues in the repeat units in the procyclic parasites, while those structures are not known in the metacyclic stage. Those polymorphisms in the composition and sequence of the sugars branching off the repeat units of the LPG have been assessed in the interaction with vertebrate and invertebrate hosts, especially for the Old World Leishmania species. However, for most of New World species of Leishmania, the role of those polymorphisms in the mmunopathology of the disease is still unknown. This study aimed to evaluate the interaction between the LPGs of two epidemiologically important Brazilian species and murine macrophages. Those include L. braziliensis and L. infantum, causative agents of cutaneous and visceral leishmaniasis, respectivelly. Mice peritoneal macrophages from BALB/c, C57BL/6, and C57BL/6 knock-out (TLR2 -/- e TLR4 -/-) lineages were primed with IFN- and stimulated with LPG from both species. Cytokine production (IL-1β, IL-2; IL-4, IL-6, IL-10, IL-12p40, IFN- and TNF-α) and nitrite concentration were determined by flow cytometry and Griess reaction, respectivelly. Western blot was performed to evaluate the activation of the MAPKs ERK and p38. Macrophages stimulated with L. braziliensis LPG, had a higher TNF-α, IL-1β, IL-6 e NO production than those stimulated with L. infantum’s. A different MAPK activation kinetics between the two species was detected. Leishmania infantum LPG exhibited a gradual activation profile until 45 min after stimulation, whereas L. braziliensis LPG showed an activation peak at 15 min. These data suggest that interpecies variations in Leishmania LPG may have an important role during initial steps of infection in the host’s innate imune system

390. Can Testing the Environment for SARS-CoV-2 Be a Signal for Staff Infections in Nursing Homes (NHs)?

Abstract Background Federal mandate requires NHs to perform weekly COVID-19 testing of staff. Testing is effective due to barriers to disclosing mild illness, but it is unclear how long the mandate will last. We explored if environmental samples can be used to signal staff COVID-19 cases as an alternative screening tool in NHs. Methods We conducted a cross sectional study to assess the value of environmental sampling as a trigger for COVID-19 testing of NH staff using data from currently performed weekly staff sweeps. We performed 35 sampling sweeps across 21 NHs from 6/2020-2/2021. For each sweep, we sampled up to 24 high touch objects in NH breakrooms (N=226), entryways (N=216), and nursing stations (N=194) assuming that positive samples were due to contamination from infected staff. Total staff and positive staff counts were tallied for the staff testing sweeps performed the week of and week prior to environmental sampling. Object samples were processed for SARS-CoV-2 using PCR (StepOnePlus) with a 1 copy/mL limit of detection. We evaluated concordance between object and staff positivity using Cohen’s kappa and calculated the positive and negative predictive value (PPV, NPV) of environmental sweeps for staff positivity, including the attributable capture of positive staff. We tested the association between the proportion of staff positivity and object contamination by room type in a linear regression model when clustering by NH. Results Among 35 environmental sweeps, 49% had SARS-CoV-2 positive objects and 69% had positive staff in the same or prior week. Mean positivity was 16% (range 0-83%) among objects and 4% (range 0-22%) among staff. Overall, NPV was 61% and Cohen’s kappa was 0.60. PPV of object sampling as an indicator of positive staff was 100% for every room type, with an attributable capture of positive staff of 76%, with values varying by room type (Table). Breakroom samples were the strongest indicator of any staff cases. Each percent increase in object positivity was associated with an increase in staff positivity in entryways (7.2% increased staff positivity, P=0.01) and nursing stations (5.7% increased staff positivity, P=0.05). Conclusion If mandatory weekly staff testing ends in NHs, environmental sampling may serve as an effective tool to trigger targeted COVID-19 testing sweeps of NH staff. Disclosures Gabrielle Gussin, MS, Medline (Other Financial or Material Support, Conducted studies in which participating hospitals and nursing homes received contributed antiseptic and cleaning products)Stryker (Sage) (Other Financial or Material Support, Conducted studies in which participating hospitals and nursing homes received contributed antiseptic products)Xttrium (Other Financial or Material Support, Conducted studies in which participating hospitals and nursing homes received contributed antiseptic products) Raveena Singh, MA, Medline (Other Financial or Material Support, Conducted studies in which participating hospitals and nursing homes received contributed antiseptic and cleaning products)Stryker (Sage) (Other Financial or Material Support, Conducted studies in which participating hospitals and nursing homes received contributed antiseptic products)Xttrium (Other Financial or Material Support, Conducted studies in which participating hospitals and nursing homes received contributed antiseptic products) Raheeb Saavedra, AS, Medline (Other Financial or Material Support, Conducted studies in which participating hospitals and nursing homes received contributed antiseptic and cleaning products)Stryker (Sage) (Other Financial or Material Support, Conducted studies in which participating hospitals and nursing homes received contributed antiseptic products)Xttrium (Other Financial or Material Support, Conducted studies in which participating hospitals and nursing homes received contributed antiseptic products) Susan S. Huang, MD, MPH, Medline (Other Financial or Material Support, Conducted studies in which participating hospitals and nursing homes received contributed antiseptic and cleaning products)Molnlycke (Other Financial or Material Support, Conducted studies in which participating hospitals and nursing homes received contributed antiseptic and cleaning products)Stryker (Sage) (Other Financial or Material Support, Conducted studies in which participating hospitals and nursing homes received contributed antiseptic and cleaning products)Xttrium (Other Financial or Material Support, Conducted studies in which participating hospitals and nursing homes received contributed antiseptic and cleaning products

Types of GIPLs.

<p>For information on M2, M3, iM2, GIPL-A, isoM3 and isoM4, see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001543#s1" target="_blank">introduction</a>. Fatty acid chains vary in different GIPL species: The predominant type fatty acid in Type-1 and Hybrid GIPLs is C_18∶0, in type-2 GIPLs the predominant lipids are C_18∶0, C_22∶0 C_24∶0 and C_26∶0. “R” in Type 1 GIPLs represent a protein linked to the GIPL structure by a ethanolamine phosphate residue (e.g. gp63 surface metalloprotease) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001543#pntd.0001543-McConville1" target="_blank">[31]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001543#pntd.0001543-Ralton1" target="_blank">[78]</a>.</p

Monosaccharide profile of <i>Leishmania</i> glycoinositolphospholipids (GIPLs).

<p>(A) Fluorophore-assisted carbohydrate electrophoresis (FACE). Lane 1, standards represented by galactose, glucose and mannose (100 µg/ml); lane 2, <i>L. braziliensis</i> GIPLs (Gb); lane 3, <i>L. infantum</i> GIPLs (Gi) and Lane 4, <i>L. donovani</i> GIPLs (Gd). (B) High performance liquid chromatography (HPLC). Gal, galactose; Glc, glucose and Man, mannose.</p

Modulation of nitrite, TNF-α and IL-12 production by <i>Leishmania</i> GIPLs in BALB/c macrophages.

<p>Cells were incubated with GIPLs (25 µg/ml) from <i>L. braziliensis</i> (Gb) <i>and L. infantum</i> (Gi) for 15 min prior to stimulation with IFN-γ (100 IU/ml) (A) or LPS (100 ng/mL) (B). Nitrite content was measured by Griess reaction; TNF-α and IL-12 concentrations were measured by ELISA. P<0.05 was considered significant. Results are the representation of three experiments.</p

Fluorophore-assisted carbohydrate electrophoresis (FACE) of <i>Leishmania</i> GIPLs.

<p>Lane 1, oligoglucose ladder represented by G2-G7; lane 2, <i>L. braziliensis</i> GIPLs (Gb) and lane 3, <i>L. infantum</i> GIPLs (Gi) and lane 4, <i>L. donovani</i> GIPLs (Gd).</p

Activation of MAPKs (ERK and p38) by <i>Leishmania</i> GIPLs in BALB/c peritoneal macrophages.

<p>Mouse peritoneal macrophages were stimulated for 30 min with 25 µg/mL of GIPLs. Dually phosphorylated MAPKs were detected by western blot. C, negative control; Gb, <i>L. braziliensis</i> GIPLs and Gi, <i>L. infantum</i> GIPLs. Also cells were incubated with GIPLs prior to stimulation with LPS; total ERK content as a normalizing protein.</p