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Germinal centres are the engines of antibody evolution. Here, using human immunodeficiency virus (HIV) Env protein immunogen priming in rhesus monkeys followed by a long period without further immunization, we demonstrate germinal centre B (B

This paper aims the study of lightweight gears through a novel approach based on a multibody model contact-based with pseudo-rigid teeth. This method considers the teeth as pseudo-rigid bodies attached to the main body through revolute joints and torsion springs with precomputed stiffness. Thanks to this approach, the lightening is considered by varying the value of the stiffness parameter. To validate the method, the first step compares the Transmission Error computed with this novel approach with the Finite Element method in quasi-static operating conditions. The second step highlights the behaviour of the Transmission Error, increasing the rotation speed.

One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity-determining regions, and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next-generation sequencing of immune repertoires, we were able to specifically identify clonal family members, synthesize the monoclonal antibodies, and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design., Description, CryoEM and next-generation sequencing were used to identify monoclonal antibodies elicited by HIV Env vaccine candidates.

Saponins are potent and safe vaccine adjuvants, but their mechanisms of action remain incompletely understood. Here, we explored the properties of several saponin formulations, including immunostimulatory complexes (ISCOMs) formed by the self-assembly of saponin and phospholipids in the absence or presence of the Toll like receptor (TLR) 4 agonist monophosphoryl lipid A (MPLA). We found that MPLA self-assembles with saponins to form particles physically resembling ISCOMs, which we termed saponin/MPLA nanoparticles (SMNP). Saponin-containing adjuvants exhibited distinctive mechanisms of action, altering lymph flow in a mast cell-dependent manner and promoting antigen entry into draining lymph nodes. SMNP was particularly effective, exhibiting even greater potency than the compositionally-related adjuvant AS01(B) in mice, and primed robust germinal center B cell, T(FH), and HIV tier 2 neutralizing antibodies in non-human primates. Altogether, these findings shed new light on mechanisms by which saponin adjuvants act to promote the immune response and suggest SMNP may be a promising adjuvant in the setting of HIV, SARS-CoV-2, and other pathogens.

SummaryGerminal centers (GCs) are the engines of antibody evolution. Using HIV Env protein immunogen priming in rhesus monkeys (RM) followed by a long period without further immunization, we demonstrate GC B cells (BGC) lasted at least 6 months (29 weeks), all the while maintaining rapid proliferation. A 186-fold BGC cell increase was present by week 10 compared to a conventional immunization. Single cell transcriptional profiling revealed that both light zone and dark zone GC states were sustained throughout the 6 months. Antibody somatic hypermutation (SHM) of BGC cells continued to accumulate throughout the 29 week priming period, with evidence of selective pressure. Additionally, Env-binding BGC cells were still 49-fold above baseline 29 weeks after immunization, suggesting that they could be active for significantly longer periods of time. High titers of HIV neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing significant immunodominance challenges for B cells, among other difficulties. Memory B cells (BMem) generated under these long priming conditions had higher levels of SHM, and both BMem cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous BGC cell lineage phylogenies spanning the >6-month GC period were identified, demonstrating continuous GC activity and selection for at least 191 days, with no additional antigen exposure. A long prime, adjuvanted, slow delivery (12-day) immunization approach holds promise for difficult vaccine targets, and suggests that patience can have great value for tuning GCs to maximize antibody responses.

This paper deals with the analysis of the mechanical efficiency of the Rzeppa transmission joint through an analytical approach. The deduction of the mechanical efficiency of this joint is not very effortless. This kind of joint is characterized by many hyperstaticities due to the numerous contacts involved. For this reason, it represents, from the simulation point of view, a very challenging task. In this paper, an analytical approach based on the deduction of the equilibrium equation of the spheres inside the joint is proposed. By solving the deduced equations, it has been possible to predict the mechanical efficiency of the joint using only a small amount of data. This approach is very powerful in the first step of the design procedure when most of the data are unknown. The results obtained by the proposed approach, have been compared with experimental data taken from the literature. The comparison demonstrates the effectiveness of the method.

Background Approximately 50% of AML patients relapse following allogeneic hematopoietic stem cell transplant therapy, leaving them with very few treatment options (Rautenberg et al. (2019) Int. J. Mol. Sci. 20:228). Rare patients who naturally develop a minor antigen-specific graft-versus-leukemia T cell response show substantially lower relapse rates (Marijt et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100:2742-2747; Spierings et al. (2013) Biol. Blood Marrow Transplant. 19:1244-1253). HA-2 (YIGEVLVSV, genotype RS_61739531 C/C or T/C) is an HLA-A*02:01- and haematopoietically-restricted minor histocompatibility antigen derived from the class I myosin protein, MYO1G (Pierce et al. (2001) J. Immunol. 167:3223-3230). Patients receiving donor lymphocyte infusion from HA-2-mismatched donors who develop HA-2-specific T cells show a graft vs leukemia response and often experience long-term remission (Marijt et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100:2742-2747), making HA-2 an ideal candidate for TCR-engineered T cell immunotherapy of liquid tumors. Methods Using TScan's proprietary ReceptorScan platform, we discovered 1,302 HA-2-specific TCRs by screening 237 million naïve CD8 + T cells from 5 healthy HA-2-negative donors. We evaluated these TCRs using our proprietary DexScan platform to select the 15 TCRs with the highest surface expression and greatest affinity for the HA-2 peptide when transferred into primary human T cells. We further tested each TCR individually in our clinical vector backbone for surface expression, selective cytotoxicity, cytokine production, and proliferation using a panel of cell lines that express varying levels of HLA-A*02:01 and MYO1G. Finally, the top 5 TCRs were evaluated for alloreactivity using an array-based screen assessing 108 MHC-I molecules individually, and for off-target cross-reactivity using our proprietary genome-wide TargetScan platform. A lead TCR with limited alloreactivity and a narrow off-target profile was selected as our lead TSC-101 TCR. The avidity of TSC-101 for its putative off-targets was further measured in peptide-pulsed experiments to better appreciate the toxicity risks associated with our lead clinical candidate. Results and Conclusion Of the 1,302 HA-2-specific TCRs identified by our ReceptorScan platform, we identified TSC-101 as the most active TCR. TSC-101 displayed no alloreactivity to 107/108 HLAs tested and limited off-target risks in a genome-wide screens. Potential off-target peptides identified for TSC-101 displayed extremely weak avidities, predicting an absence of toxicity risks for our clinical candidate. Based on these results, TSC-101 has been advanced to IND-enabling activities to prepare for first-in-human testing in 2022. To our knowledge, this is the first clinical grade HA-2-specifc TCR being developed for immunotherapy for liquid tumors. Disclosures Macbeath: TScan Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Following immunization, high affinity antibody responses develop within germinal centers (GCs), specialized sites within follicles of the lymph node (LN) where B cells proliferate and undergo somatic hypermutation. Antigen availability within GCs is important, as B cells must acquire and present antigen to follicular helper T cells to drive this process. However, recombinant protein immunogens such as soluble HIV envelope (Env) trimers do not efficiently accumulate in follicles following traditional immunization. Here we demonstrate two strategies to concentrate HIV Env immunogens in follicles, via the formation of immune complexes (ICs) or by employing self-assembling protein nanoparticles for multivalent display of Env antigens. Using rhesus macaques, we show that within a few days following immunization, free trimers were present in a diffuse pattern in draining LNs, while trimer ICs and Env nanoparticles accumulated in B cell follicles. Whole LN imaging strikingly revealed that ICs and trimer nanoparticles concentrated in as many as 500 follicles in a single lymph node within 2 days after immunization. Imaging of LNs collected 7 days post-immunization showed that Env nanoparticles persisted on follicular dendritic cells in the light zone of nascent germinal centers. These findings suggest that the form of antigen administered in vaccination can dramatically impact localization in lymphoid tissues and provides a new rationale for the enhanced immune responses observed following immunization with immune complexes or nanoparticles.

Following immunization, high-affinity antibody responses develop within germinal centers (GCs), specialized sites within follicles of the lymph node (LN) where B cells proliferate and undergo somatic hypermutation. Antigen availability within GCs is important, as B cells must acquire and present antigen to follicular helper T cells to drive this process. However, recombinant protein immunogens such as soluble human immunodeficiency virus (HIV) envelope (Env) trimers do not efficiently accumulate in follicles following traditional immunization. Here, we demonstrate two strategies to concentrate HIV Env immunogens in follicles, via the formation of immune complexes (ICs) or by employing self-assembling protein nanoparticles for multivalent display of Env antigens. Using rhesus macaques, we show that within a few days following immunization, free trimers were present in a diffuse pattern in draining LNs, while trimer ICs and Env nanoparticles accumulated in B cell follicles. Whole LN imaging strikingly revealed that ICs and trimer nanoparticles concentrated in as many as 500 follicles in a single LN within two days after immunization. Imaging of LNs collected seven days postimmunization showed that Env nanoparticles persisted on follicular dendritic cells in the light zone of nascent GCs. These findings suggest that the form of antigen administered in vaccination can dramatically impact localization in lymphoid tissues and provides a new rationale for the enhanced immune responses observed following immunization with ICs or nanoparticles.

SUMMARYThe observation that humans can produce broadly neutralizing antibodies (bnAbs) against HIV-1 has generated enthusiasm about the potential for a bnAb vaccine against HIV-1. Conventional immunization strategies will likely be insufficient for the development of a bnAb HIV vaccine and vaccines to other difficult pathogens, due to the significant immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. Using longitudinal lymph node fine needle aspirates, we found that two independent methods of slow delivery immunization of rhesus macaques (RM) resulted in larger GCs, more robust and sustained GC Tfh cell responses, and GC B cells with improved Env-binding, which correlated with the development of ~20 to 30-fold higher titers of tier 2 HIV-1 nAbs. Using a new RM genomic immunoglobulin loci reference sequence, we identified differential IgV gene usage between slow delivery immunized and conventional bolus immunized animals. The most immunodominant IgV gene used by conventionally immunized animals was associated with many GC B cell lineages. Ab mapping of those GC B cell specificities demonstrated targeting of an immunodominant non-neutralizing trimer base epitope, while that response was muted in slow delivery immunized animals. Thus, alternative immunization strategies appear to enhance nAb development by altering GCs and modulating immunodominance of non-neutralizing epitopes.

The Lewis rat is the only known warm-blooded animal that has sterile immunity toToxoplasma. Upon invasion of Lewis rat macrophagesToxoplasmarapidly activates the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 1 (NLRP1) inflammasome resulting in interleukin (IL)-1β secretion and a form of cell death known as pyroptosis, which preventsToxoplasmareplication. Using a chemical mutagenesis screen we identifiedToxoplasmamutants that no longer induced pyroptosis. Whole genome sequencing led to the identification of threeToxoplasmaparasitophorous vacuole-localized dense granule proteins, GRA35, GRA42 and GRA43 that are individually required for inflammasome activation in Lewis rat macrophages. Macrophage infection with Δgra35, Δgra42, and Δgra43parasites leads to greatly reduced cell death and reduced IL-1β secretion. Lewis rat macrophage infected with parasites containing single, double or triple deletion of these GRAs showed similar levels of cell viability suggesting the three GRAs function in the same pathway that activates the inflammasome. Deletion ofGRA42andGRA43resulted in GRA35, and other GRAs, being retained inside the parasitophorous vacuole instead of being localized to the parasitophorous vacuole membrane.Toxoplasmadeficient in GRA35, GRA42 or GRA43 do not establish chronic infection in Lewis rats, but have reduced cyst number in parasite-susceptible F344 rats, in whichToxoplasmadoes not activate the NLRP1 inflammasome, revealing these GRAs determine parasitein vivofitness independent of their role in inflammasome activation. Overall, our data suggest thatToxoplasmadense granule proteins that localize to the parasitophorous vacuole membrane are novel mediators of host NLRP1 inflammasome activation.ImportanceInflammasomes are a major component of the innate immune system and responsible for detecting various microbial and environmental danger signals. The Lewis rat has sterile immunity toToxoplasmabecause upon invasion of Lewis rat macrophages the parasite rapidly activates the NLRP1 inflammasome resulting in cell death and parasite elimination. The work reported here identified thatToxoplasmaGRA35, GRA42 and GRA43 are required for activation of the Lewis rat NLRP1 inflammasome. GRA42 and GRA43 mediate the correct localization of other GRAs, including GRA35, to the parasitophorous vacuole membrane. In addition to their role in inflammasome activation, these three GRAs are also important for parasitein vivofitness in aToxoplasma-susceptible rat strain. Thus, these results give new insight into NLRP1 inflammasome activation byToxoplasmaeffectors and identified three GRAs that are required for pathogenesis of the parasite.

Conventional immunization strategies will likely be insufficient for the development of a broadly neutralizing antibody (bnAb) vaccine for HIV or other difficult pathogens because of the immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. We found that two independent methods of slow delivery immunization of rhesus monkeys (RMs) resulted in more robust T follicular helper (TFH) cell responses and GC B cells with improved Env-binding, tracked by longitudinal fine needle aspirates. Improved GCs correlated with the development of >20-fold higher titers of autologous nAbs. Using a new RM genomic immunoglobulin locus reference, we identified differential IgV gene use between immunization modalities. Ab mapping demonstrated targeting of immunodominant non-neutralizing epitopes by conventional bolus-immunized animals, whereas slow delivery-immunized animals targeted a more diverse set of epitopes. Thus, alternative immunization strategies can enhance nAb development by altering GCs and modulating the immunodominance of non-neutralizing epitopes. An integrated immunological, bioinformatic and imaging approach demonstrates how slow delivery immunization enhances neutralizing antibody and germinal center reactions over conventional strategies in response to HIV Env protein immunization in non-human primates., National Institutes of Health (Grant AI125068), National Institutes of Health (Grant Al100663)

Skeletal muscle atrophy is a debilitating condition that can arise due to aging, cancer, corticosteroid use, and denervation. To better characterize the molecular genetic events of neurogenic atrophy, a previous study analyzed gene expression patterns in gastrocnemius muscle following sciatic nerve transection and found for the first time that Zinc Finger Protein 593 (Zfp593) is expressed in skeletal muscle and is induced in response to denervation. Quantitative PCR and Western blot analyses confirmed that Zfp593 is expressed in both proliferating myoblasts and differentiated myotubes. To assess sub-cellular location, GFP-tagged Zfp593 was expressed in C2C12 cells and found to localize to the nucleus. The Zfp593 protein possesses a putative zinc finger domain and is believed to function as a modulator of the Oct-2 transcription factor. Interestingly, ectopic expression of Zfp593 did not affect the ability of Oct-1 or Oct-2 to inhibit an Oct reporter gene in muscle cells. Finally, Zfp593 overexpression in cultured muscle cells resulted in significant repression of muscle cell differentiation and attenuation of ERK1/2 and p38 phosphorylation, but did not vitiate protein synthesis. The discovery that Zfp593 is expressed in skeletal muscle combined with the observation that it is induced in response to neurogenic atrophy furthers our understanding of the molecular genetic events of muscle wasting.

The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells.

© 2015 Elsevier Inc. Toxoplasma gondii is a protozoan pathogen in the phylum Apicomplexa that resides within an intracellular parasitophorous vacuole (PV) that is selectively permeable to small molecules through unidentified mechanisms. We have identified GRA17 as a Toxoplasma-secreted protein that localizes to the parasitophorous vacuole membrane (PVM) and mediates passive transport of small molecules across the PVM. GRA17 is related to the putative Plasmodium translocon protein EXP2 and conserved across PV-residing Apicomplexa. The PVs of GRA17-deficient parasites have aberrant morphology, reduced permeability to small molecules, and structural instability. GRA17-deficient parasites proliferate slowly and are avirulent in mice. These GRA17-deficient phenotypes are rescued by complementation with Plasmodium EXP2. GRA17 functions synergistically with a related protein, GRA23. Exogenous expression of GRA17 or GRA23 alters the membrane conductance properties of Xenopus oocytes in a manner consistent with a large non-selective pore. Thus, GRA17 and GRA23 provide a molecular basis for PVM permeability and nutrient access.

We present the activities of the "New Physics" working group for the "Physics at TeV Colliders" workshop (Les Houches, France, 30 May-17 June, 2011). Our report includes new agreements on formats for interfaces between computational tools, new tool developments, important signatures for searches at the LHC, recommendations for presentation of LHC search results, as well as additional phenomenological studies.

Induction of immunity that limits Toxoplasma gondii infection in mice is critically dependent on the activation of the innate immune response. In this study, we investigated the role of cytoplasmic nucleotide-binding domain and leucine-rich repeat containing a pyrin domain (NLRP) inflammasome sensors during acute toxoplasmosis in mice. We show that in vitro Toxoplasma infection of murine bone marrow-derived macrophages activates the NLRP3 inflammasome, resulting in the rapid production and cleavage of interleukin-1β (IL-1β), with no measurable cleavage of IL-18 and no pyroptosis. Paradoxically, Toxoplasma-infected mice produced large quantities of IL-18 but had no measurable IL-1β in their serum. Infection of mice deficient in NLRP3, caspase-1/11, IL-1R, or the inflammasome adaptor protein ASC led to decreased levels of circulating IL-18, increased parasite replication, and death. Interestingly, mice deficient in NLRP1 also displayed increased parasite loads and acute mortality. Using mice deficient in IL-18 and IL-18R, we show that this cytokine plays an important role in limiting parasite replication to promote murine survival. Our findings reveal T. gondii as a novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host resistance to toxoplasmosis. IMPORTANCE Inflammasomes are multiprotein complexes that are a major component of the innate immune system. They contain “sensor” proteins that are responsible for detecting various microbial and environmental danger signals and function by activating caspase-1, an enzyme that mediates cleavage and release of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Toxoplasma gondii is a highly successful protozoan parasite capable of infecting a wide range of host species that have variable levels of resistance. We report here that T. gondii is a novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host resistance to toxoplasmosis. Using mice deficient in IL-18 and IL-18R, we show that the IL-18 cytokine plays a pivotal role by limiting parasite replication to promote murine survival., National Institutes of Health (U.S.) (Intramural Research Program of the NIH and NIAID), Crohn's and Colitis Foundation of America (Research Fellowship), Crohn's and Colitis Foundation of America (CCFA Helmsley Scholar), National Institutes of Health (U.S.) (NIH grant AI104170), National Institutes of Health (U.S.) (R01-AI080621), Pew Charitable Trusts (Pew Scholars Program in the Biomedical Sciences), Canadian Institute for Advanced Research (CIFAR Program for Integrated Microbial Biodiversity)

Osteopontin (OPN) is a proinflammatory cytokine produced by T-cells, macrophages, osteoclasts, and several other cell types, which confers immunity to many intracellular pathogens. OPN was first identified as an early marker of cellular activation of T-lymphocytes and subsequently was shown to play a role in cancer through its ability to promote cell survival and inflammation. OPN levels are elevated in the plasma and cerebrospinal fluid of HIV-infected individuals and even more so in those suffering from HIV-related neurocognitive impairment. The infiltration of monocytes and macrophages both infected and uninfected into the brain is the first step in HIV pathogenesis of the central nervous system. Inhibition of OPN in macrophages significantly impairs HIV replication. In an effort to identify and understand the role of OPN in the neuropathogenesis of HIV infection, we are using a combination of in vitro, ex vivo and in vivo approaches. In this study we have used a molecular approach and a surrogate cell culture model to identify the domains of OPN that are required to enhance HIV replication. We found that N- and C-terminal fragments, encoding multiple motifs including sequences involved in binding integrins and CD44, a domain know to promote adhesion contribute to OPN’s ability to increase HIV replication. Use of inhibitors against c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase (PI-3K) impaired the ability of OPN to increase the integrin subunit 1 or CD29 on the surface of HIV-infected and bystander cells. These results suggest that multiple OPN-regulated cellular pathways are commandeered by HIV to promote productive replication and cell-to-cell spread.

Hypoplasia of the distal abdominal aorta, first described by Quain in 1847, is rarely associated with atherosclerotic occlusive disease. The condition usually occurs early in life; the principal clinical features are hypertension and arterial insufficiency of the lower extremities which is often well tolerated. Some 20 patients had operations for abdominal aortic hypoplasia; 16 underwent prosthetic reconstruction and four had aortoiliac endarterectomy with angioplasty. Reconstructive techniques were influenced by narrow renal arteries and by the presence of extensive atheroma. The long-term prognosis is often poor, especially in patients with extensive disease.

Echocardiographic studies were performed in 15 patients, aged 8 months to 20 years, with congenital valvular aortic stenosis confirmed by hemodynamic findings. Seven patients showed an anomalous pattern of the valvular echo in diastole (multiple valve echoes), seven exhibited an eccentric closure, and only in two patients the area was significantly reduced in systole. Therefore aortic valve diastolic pattern was more important than systolic appearance in the echocardiografic diagnosis of congenital valvular aortic stenosis. In all cases a correlation was found between the left ventricular pressure obtained with this non invasive method and the pressure measured in the left ventricle at the cardiac catheterization (p less than 0.001).

A case of gall bladder haematoma observed echographically in a dialysed patient is reported. The observation was probably related to the administration of anticoagulants during dialysis.

Ultrasound during pregnancy is a valid method to determine foetal growth. Occasionally abnormalities are detected. We report a case in which an echographic study showed a placental chorioangioma. We underline ultrasound patterns and examine clinical features.