Slow Delivery Immunization Enhances HIV Neutralizing Antibody and Germinal Center Responses via Modulation of Immunodominance.

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.

Selection of internalizing RNA aptamers into human breast cancer cells derived from primary sites.

Breast cancer is the most common cancer in women. Although chemotherapy is still broadly used in its treatment, adverse effects remain a challenge. In this scenario, aptamers emerge as a promising alternative for theranostic applications. Studies using breast cancer cell lines provide useful information in laboratory and preclinical investigations, most of which use cell lines established from metastatic sites. However, these cell lines correspond to cell populations of the late stage of tumor progression. On the other hand, studies using breast cancer cells established from primary sites make it possible to search for new theranostic approaches in the early stages of the disease. Therefore, this work aimed to select RNA aptamers internalized by MGSO-3 cells, a human breast cancer cell line, derived from a primary site previously established in our laboratory. Using the Cell-Internalization SELEX method, we have selected two candidate aptamers (ApBC1 and ApBC2). We evaluated their internalization efficiencies, specificities, cellular localization by Reverse Transcription-qPCR (RT-qPCR) and confocal microscopy assays. The results suggest that both aptamers were efficiently internalized by human breast cancer cells, MACL-1, MDA-MB-231, and especially by MGSO-3 cells. Furthermore, both aptamers could effectively distinguish human breast cancer cells derived from normal human mammary cell (MCF 10A) and prostate cancer cell (PC3) lines. Therefore, ApBC1 and ApBC2 could be promising candidate molecules for theranostic applications, even in the early stages of tumor progression.

Cytoskeletal tension actively sustains the migratory T-cell synaptic contact.

When migratory T cells encounter antigen-presenting cells (APCs), they arrest and form radially symmetric, stable intercellular junctions termed immunological synapses which facilitate exchange of crucial biochemical information and are critical for T-cell immunity. While the cellular processes underlying synapse formation have been well characterized, those that maintain the symmetry, and thereby the stability of the synapse, remain unknown. Here we identify an antigen-triggered mechanism that actively promotes T-cell synapse symmetry by generating cytoskeletal tension in the plane of the synapse through focal nucleation of actin via Wiskott-Aldrich syndrome protein (WASP), and contraction of the resultant actin filaments by myosin II. Following T-cell activation, WASP is degraded, leading to cytoskeletal unraveling and tension decay, which result in synapse breaking. Thus, our study identifies and characterizes a mechanical program within otherwise highly motile T cells that sustains the symmetry and stability of the T cell-APC synaptic contact.

Comparison of skeletal muscle decellularization protocols and recellularization with adipose-derived stem cells for tissue engineering.

Decellularization is a novel technique employed for scaffold manufacturing, as a strategy for skeletal muscle (SM) tissue engineering applications. However, poor decellularization efficacy is still a problem for the use of decellularized scaffolds as truly biocompatible biomaterials. For recellularization, adipose-derived stem cells (ASCs) are a good option, due to their immunomodulatory and pro-regenerative capacity, but few studies have described their combination with muscle-decellularized matrices (mDMs). This work aimed to evaluate the efficiency of four multi-step decellularization protocols to produce mDMs and to investigate in vitro biocompatibility with ASCs. Here, we described the different efficacies of muscle decellularization methods, suggesting the need for stricter standardization of the method, considering the large range of applications in SM tissue engineering, which is also a promising platform for preclinical studies with rat disease models using autologous cells.

STING agonist delivery by tumour-penetrating PEG-lipid nanodiscs primes robust anticancer immunity.

Activation of the innate immune STimulator of INterferon Genes (STING) pathway potentiates antitumour immunity, but systemic delivery of STING agonists to tumours is challenging. We conjugated STING-activating cyclic dinucleotides (CDNs) to PEGylated lipids (CDN-PEG-lipids; PEG, polyethylene glycol) via a cleavable linker and incorporated them into lipid nanodiscs (LNDs), which are discoid nanoparticles formed by self-assembly. Compared to state-of-the-art liposomes, intravenously administered LNDs carrying CDN-PEG-lipid (LND-CDNs) exhibited more efficient penetration of tumours, exposing the majority of tumour cells to STING agonist. A single dose of LND-CDNs induced rejection of established tumours, coincident with immune memory against tumour rechallenge. Although CDNs were not directly tumoricidal, LND-CDN uptake by cancer cells correlated with robust T-cell activation by promoting CDN and tumour antigen co-localization in dendritic cells. LNDs thus appear promising as a vehicle for robust delivery of compounds throughout solid tumours, which can be exploited for enhanced immunotherapy.

DNA aptamers selection for Staphylococcus aureus cells by SELEX and Cell-SELEX.

BACKGROUND: Staphylococcus aureus is the most common bacteria found in skin, soft tissues, bone, and bone prostheses infections. The aim of this study was to select DNA aptamers for S. aureus to be applied in the diagnosis of bacteria. METHODS AND RESULTS: We used SELEX (Systematic Evolution of Ligands by EXponencial Enrichment) for peptidoglycan followed by cell-SELEX with S. aureus cells as target. Four sequences showed significantly higher binding to S. aureus distinguishing it from the control cells of other significant microbial species: Escherichia coli, Candida albicans, Streptococcus pyogenes and Streptococcus pneumoniae. In particular, ApSA1 (Kd = 62.7 ± 5.6 nM) and ApSA3 (Kd = 43.3 ± 3.0 nM) sequences combined high affinity and specificity for S. aureus, considering all microorganisms tested. CONCLUSIONS: Our results demonstrated that these aptamers were able to identify peptidoglycan in the S. aureus surface and have great potential for use in the development of radiopharmaceuticals capable to identify S. aureus infectious foci, as well as in other aptamer-based methodologies for bacteria diagnosis.

Controlled Lipid Self-Assembly for Scalable Manufacturing of Next-Generation Immune Stimulating Complexes.

Immune stimulating complexes (ISCOMs) are safe and effective saponin-based adjuvants formed by the self-assembly of saponin, cholesterol, and phospholipids in water to form cage-like 30-40 nm diameter particles. Inclusion of the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) in ISCOM particles yields a promising next-generation adjuvant termed Saponin-MPLA NanoParticles (SMNP). In this work, we detail protocols to produce ISCOMs or SMNP via a tangential flow filtration (TFF) process suitable for scalable synthesis and Good Manufacturing Practice (GMP) production of clinical-grade adjuvants. SMNP or ISCOM components were solubilized in micelles of the surfactant MEGA-10, then diluted below the critical micelle concentration (CMC) of the surfactant to drive ISCOM self-assembly. Assembly of ISCOM/SMNP particles using the purified saponin QS-21 used in clinical-grade saponin adjuvants was found to require controlled stepwise dilution of the initial micellar solution, to prevent formation of undesirable kinetically-trapped aggregate species. An optimized protocol gave yields of ~77% based on the initial feed of QS-21 and the final SMNP particle composition mirrored the feed ratios of the components. Further, samples were highly homogeneous with comparable quality to that of material prepared at lab scale by dialysis and purified via size-exclusion chromatography. This protocol may be useful for clinical preparation of ISCOM-based vaccine adjuvants and therapeutics.

Comparison of direct MRI guided versus atlas-based targeting for subthalamic nucleus and globus pallidus deep brain stimulation.

PURPOSE: The subthalamic nucleus (STN) and globus pallidus internus (GPi) targets for deep brain stimulation (DBS) can be defined by atlas coordinates or direct visualisation of the target on MRI. The aim of this study was to evaluate geometric differences between atlas-based targeting and MRI-guided direct targeting. METHODS: One-hundred-nine Parkinson's disease or dystonia patients records who underwent DBS surgery between 2005 and 2016 were prospectively reviewed. MRI-guided direct targeting coordinates was used to implant 205 STN and 64 GPi electrodes and compared with atlas-based coordinates. RESULTS: The directly targeted coordinates (mean, SD, range) for STN were x: [9.9 ± 1.1 (7.1 - 13.2)], y: [-0.8 ± 1.1 (-4.2 - 2)] and z: [-4.7 ± 0.53 (-5.9 - -3.2)]. The mean value for the STN was 2.1 mm more medial (p < 0.0001), 1.2 mm more anterior (p < 0.0001) and 0.7 mm more ventral (p < 0.0001) than the atlas target. The targeted coordinates for GPi were x: [22.3 ± 2.0 (17.8 - 26.1)], y: [-0.2 ± 2.2 (-4.5 - 3.4)], z: [-4.3 ± 0.8 (-6.2 - -2.3)]. The mean value for the GPi was 2.2 mm (p < 0.001) more posterior and 0.3 mm (p < 0.01) more ventral than the atlas-based coordinates. CONCLUSION: MRI-guided targeting may be more accurate than atlas-based targeting due to individual variations in anatomy.

Human adipose-derived stromal/stem cells are distinct from dermal fibroblasts as evaluated by biological characterization and RNA sequencing.

Human adipose-derived stromal/stem cells (ASC) have immunomodulatory properties and the potential to differentiate into several cell lines, important for application in regenerative medicine. However, the contamination with dermal fibroblasts (FIB) can impair the beneficial effects of ASC in cell therapy. It is then essential to develop new strategies that contribute to the distinction between these two cell types. In this study, we performed functional assays, high-throughput RNA sequencing (RNA-Seq) and quantitative PCR (qPCR) to find new markers that can distinguish ASC and FIB. We showed that ASC have adipogenic and osteogenic differentiation capacity and alkaline phosphatase activity, not observed in FIB. Gene expression variation analysis identified more than 2000 differentially expressed genes (DEG) between these two cell types. We validated 16 genes present in the list of DEG, including the alkaline phosphatase gene (ALPL). In conclusion, we showed that ASC and FIB have distinct biological properties as demonstrated by alkaline phosphatase activity and differentiation capacity, besides having different gene expression profiles. SIGNIFICANCE OF THE STUDY: Although many differences between stromal stem cells derived from human adipose tissue (ASC) and human dermal fibroblasts (FIB) are described, it is still difficult to find specific markers to differentiate them. This problem can interfere with the therapeutic use of ASC. This work aimed to find new markers to differentiate these two cell populations. Our findings suggest that these cells can be distinguished by biological and molecular characteristics, such as adipogenic and osteogenic differentiation, alkaline phosphatase activity and differential gene expression profiles. The DEG were related to the regulation of the cell cycle, development process, structural organization of the cell and synthesis of the extracellular matrix. This study helps to find new cellular markers to distinguish the two populations and to better understand the properties of these cells, which can improve cell therapy.

DNA aptamers selection for carcinoembryonic antigen (CEA).

Carcinoembryonic antigen (CEA) is a glycoprotein antigen generally used for diagnosis, prognosis and treatment monitoring of several types of tumors, including colorectal cancer. Nucleic acid aptamers are DNA or RNA oligonucleotides capable of binding with high specificity and affinity to a molecular target. The aim of this study was to obtain aptamers specific to CEA for use as radiopharmaceuticals in colorectal cancer diagnosis. Five aptamers were selected through the Systematic Evolution of Ligands by EXponencial Enrichment (SELEX) and tested using T84 (CEA+) and Hela (CEA-) cells. Apta 3 and Apta 5 showed the best results presenting high specificity and affinity for T84 cells, with dissociation constants (Kd) of 60.4 ± 5.7 nM and 37.8 ± 5.8 nM, respectively. These results indicate that Apta 3 and Apta 5 are promising candidates for identifying tumor cells that overexpress CEA.

Immunogenicity of RNA Replicons Encoding HIV Env Immunogens Designed for Self-Assembly into Nanoparticles.

RNA replicons are a promising platform technology for vaccines. To evaluate the potential of lipid nanoparticle-formulated replicons for delivery of HIV immunogens, we designed and tested an alphavirus replicon expressing a self-assembling protein nanoparticle immunogen, the glycoprotein 120 (gp120) germline-targeting engineered outer domain (eOD-GT8) 60-mer. The eOD-GT8 immunogen is a germline-targeting antigen designed to prime human B cells capable of evolving toward VRC01-class broadly neutralizing antibodies. Replicon RNA was encapsulated with high efficiency in 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-based lipid nanoparticles, which provided effective delivery in the muscle and expression of luciferase lasting ∼30 days in normal mice, contrasting with very brief and low levels of expression obtained by delivery of equivalent modified mRNA (modRNA). eOD-GT8 60-mer-encoding replicons elicited high titers of gp120-specific antibodies following a single injection in mice, and increased levels of antigen-specific germinal center B cells compared with protein immunization. Immunization of transgenic mice expressing human inferred-germline VRC01 heavy chain B cell receptors that are the targets of the eOD antigen led to priming of B cells and somatic hypermutation consistent with VRC01-class antibody development. Altogether, these data suggest replicon delivery of Env immunogens may be a promising avenue for HIV vaccine development.

Sustained antigen availability during germinal center initiation enhances antibody responses to vaccination.

Natural infections expose the immune system to escalating antigen and inflammation over days to weeks, whereas nonlive vaccines are single bolus events. We explored whether the immune system responds optimally to antigen kinetics most similar to replicating infections, rather than a bolus dose. Using HIV antigens, we found that administering a given total dose of antigen and adjuvant over 1-2 wk through repeated injections or osmotic pumps enhanced humoral responses, with exponentially increasing (exp-inc) dosing profiles eliciting >10-fold increases in antibody production relative to bolus vaccination post prime. Computational modeling of the germinal center response suggested that antigen availability as higher-affinity antibodies evolve enhances antigen capture in lymph nodes. Consistent with these predictions, we found that exp-inc dosing led to prolonged antigen retention in lymph nodes and increased Tfh cell and germinal center B-cell numbers. Thus, regulating the antigen and adjuvant kinetics may enable increased vaccine potency.

Translocation of Epidermal Growth Factor (EGF) to the nucleus has distinct kinetics between adipose tissue-derived mesenchymal stem cells and a mesenchymal cancer cell lineage.

Nuclear Epidermal Growth Factor Receptor (EGFR) has been associated with worse prognosis and treatment resistance for several cancer types. After Epidermal Growth Factor (EGF) binding, the ligand-receptor complex can translocate to the nucleus where it functions in oncological processes. By three-dimensional quantification analysis of super-resolution microscopy images, we verified the translocation kinetics of fluorescent conjugated EGF to the nucleus in two mesenchymal cell types: human adipose tissue-derived stem cells (hASC) and SK-HEP-1 tumor cells. The number of EGF clusters in the nucleus does not change after 10 min of stimulation with EGF in both cells. The total volume occupied by EGF clusters in the nucleus of hASC also does not change after 10 min of stimulation with EGF. However, the total volume of EGF clusters increases only after 20 min in SK-HEP-1 cells nuclei. In these cells the nuclear volume occupied by EGF is 3.2 times higher than in hASC after 20 min of stimulation, indicating that translocation kinetics of EGF differs between these two cell types. After stimulation, EGF clusters assemble in larger clusters in the cell nucleus in both cell types, which suggests specific sub-nuclear localizations of the receptor. Super-resolution microscopy images show that EGF clusters are widespread in the nucleoplasm, and can be localized in nuclear envelope invaginations, and in the nucleoli. The quantitative study of EGF-EGFR complex translocation to the nucleus may help to unravel its roles in health and pathological conditions, such as cancer.

Vestibular and oculomotor influences on visual dependency.

The degree to which a person relies on visual stimuli for spatial orientation is termed visual dependency (VD). VD is considered a perceptual trait or cognitive style influenced by psychological factors and mediated by central reweighting of the sensory inputs involved in spatial orientation. VD is often measured with the rod-and-disk test, in which participants align a central rod to the subjective visual vertical (SVV) in the presence of a background that is either stationary or rotating around the line of sight-dynamic SVV. Although this task has been employed to assess VD in health and vestibular disease, what effect torsional nystagmic eye movements may have on individual performance is unknown. Using caloric ear irrigation, 3D video-oculography, and the rod-and-disk test, we show that caloric torsional nystagmus modulates measures of VD and demonstrate that increases in tilt after irrigation are positively correlated with changes in ocular torsional eye movements. When the direction of the slow phase of the torsional eye movement induced by the caloric is congruent with that induced by the rotating visual stimulus, there is a significant increase in tilt. When these two torsional components are in opposition, there is a decrease. These findings show that measures of VD can be influenced by oculomotor responses induced by caloric stimulation. The findings are of significance for clinical studies, as they indicate that VD, which often increases in vestibular disorders, is modulated not only by changes in cognitive style but also by eye movements, in particular nystagmus.

Transcriptional analysis of murine macrophages infected with different Toxoplasma strains identifies novel regulation of host signaling pathways.

Most isolates of Toxoplasma from Europe and North America fall into one of three genetically distinct clonal lineages, the type I, II and III lineages. However, in South America these strains are rarely isolated and instead a great variety of other strains are found. T. gondii strains differ widely in a number of phenotypes in mice, such as virulence, persistence, oral infectivity, migratory capacity, induction of cytokine expression and modulation of host gene expression. The outcome of toxoplasmosis in patients is also variable and we hypothesize that, besides host and environmental factors, the genotype of the parasite strain plays a major role. The molecular basis for these differences in pathogenesis, especially in strains other than the clonal lineages, remains largely unexplored. Macrophages play an essential role in the early immune response against T. gondii and are also the cell type preferentially infected in vivo. To determine if non-canonical Toxoplasma strains have unique interactions with the host cell, we infected murine macrophages with 29 different Toxoplasma strains, representing global diversity, and used RNA-sequencing to determine host and parasite transcriptomes. We identified large differences between strains in the expression level of known parasite effectors and large chromosomal structural variation in some strains. We also identified novel strain-specifically regulated host pathways, including the regulation of the type I interferon response by some atypical strains. IFNß production by infected cells was associated with parasite killing, independent of interferon gamma activation, and dependent on endosomal Toll-like receptors in macrophages and the cytoplasmic receptor retinoic acid-inducible gene 1 (RIG-I) in fibroblasts.

Admixture and recombination among Toxoplasma gondii lineages explain global genome diversity.

Toxoplasma gondii is a highly successful protozoan parasite that infects all warm-blooded animals and causes severe disease in immunocompromised and immune-naïve humans. It has an unusual global population structure: In North America and Europe, isolated strains fall predominantly into four largely clonal lineages, but in South America there is great genetic diversity and the North American clonal lineages are rarely found. Genetic variation between Toxoplasma strains determines differences in virulence, modulation of host-signaling pathways, growth, dissemination, and disease severity in mice and likely in humans. Most studies on Toxoplasma genetic variation have focused on either a few loci in many strains or low-resolution genome analysis of three clonal lineages. We use whole-genome sequencing to identify a large number of SNPs between 10 Toxoplasma strains from Europe and North and South America. These were used to identify haplotype blocks (genomic regions) shared between strains and construct a Toxoplasma haplotype map. Additional SNP analysis of RNA-sequencing data of 26 Toxoplasma strains, representing global diversity, allowed us to construct a comprehensive genealogy for Toxoplasma gondii that incorporates sexual recombination. These data show that most current isolates are recent recombinants and cannot be easily grouped into a limited number of haplogroups. A complex picture emerges in which some genomic regions have not been recently exchanged between any strains, and others recently spread from one strain to many others.

The rhoptry proteins ROP18 and ROP5 mediate Toxoplasma gondii evasion of the murine, but not the human, interferon-gamma response.

The obligate intracellular parasite Toxoplasma gondii secretes effector proteins into the host cell that manipulate the immune response allowing it to establish a chronic infection. Crosses between the types I, II and III strains, which are prevalent in North America and Europe, have identified several secreted effectors that determine strain differences in mouse virulence. The polymorphic rhoptry protein kinase ROP18 was recently shown to determine the difference in virulence between type I and III strains by phosphorylating and inactivating the interferon-γ (IFNγ)-induced immunity-related GTPases (IRGs) that promote killing by disrupting the parasitophorous vacuole membrane (PVM) in murine cells. The polymorphic pseudokinase ROP5 determines strain differences in virulence through an unknown mechanism. Here we report that ROP18 can only inhibit accumulation of the IRGs on the PVM of strains that also express virulent ROP5 alleles. In contrast, specific ROP5 alleles can reduce IRG coating even in the absence of ROP18 expression and can directly interact with one or more IRGs. We further show that the allelic combination of ROP18 and ROP5 also determines IRG evasion and virulence of strains belonging to other lineages besides types I, II and III. However, neither ROP18 nor ROP5 markedly affect survival in IFNγ-activated human cells, which lack the multitude of IRGs present in murine cells. These findings suggest that ROP18 and ROP5 have specifically evolved to block the IRGs and are unlikely to have effects in species that do not have the IRG system, such as humans.

Enhancing Ophthalmic Triage: identification of new clinical features to support healthcare professionals in triage.

OBJECTIVE: To investigate which features from a patient's history are either high or low risk that could support healthcare professionals in ophthalmic emergency triage. METHODS: Prospective, 12,584 visits from 11,733 adult patients attending an Accident and Emergency department at a single tertiary centre were analysed. Data were collected by ophthalmic nurses working in triage, using an online form from August 2021 to April 2022. Multivariate analysis (MVA) was conducted to identify which features from the patients' history would be associated with emergency care. RESULTS: This study found that 45.5% (5731 patient visits (PV)) required a same day eye emergency examination (SDEE), 11.3% (1416 PV) needed urgent care, and 43.2% (5437 PV) were appropriate for elective consultations with a GP or optometrist. The MVA top ten features that were statistically significant (p < 0.05) that would warrant SDEE with odds ratio (95% CI) were: bilateral eye injury 36.5 [15.6-85.5], unilateral eye injury 25.8 [20.9-31.7], vision loss 4.8 [2.9-7.8], post-operative ophthalmic ( < 4 weeks) 4.6 [3.8-5.7], contact lens wearer 3.9 [3.3-4.7], history of uveitis 3.9 [3.3-4.7], photophobia 2.9 [2.4-3.6], unilateral dark shadow/curtain in vision 2.4 [1.8-3.0], unilateral injected red eye 2.0 [1.8-2.2] and rapid change in visual acuity 1.8 [1.5-2.2]. CONCLUSION: This study characterises presenting features covering almost 100 ophthalmic acute presentations that are commonly seen in emergency and elective care. This information could supplement current red flag indicators and support healthcare professionals in ophthalmic triage. Further research is required to evaluate the cost effectivity and safety of our findings for triaging acute presentations.

"Target-and-release" nanoparticles for effective immunotherapy of metastatic ovarian cancer.

Immunotherapies such as checkpoint inhibitors (CPI) are effective in treating several advanced cancers, but these treatments have had limited success in metastatic ovarian cancer (OC). Here, we engineered liposomal nanoparticles (NPs) carrying a layer-by-layer (LbL) polymer coating that promotes their binding to the surface of OC cells. Covalent anchoring of the potent immunostimulatory cytokine interleukin-12 (IL-12) to phospholipid headgroups of the liposome core enabled the LbL particles to concentrate IL-12 in disseminated OC tumors following intraperitoneal administration. Shedding of the LbL coating and serum protein-mediated extraction of IL-12-conjugated lipids from the liposomal core over time enabled IL-12 to disseminate in the tumor bed following rapid NP localization in tumor nodules. Optimized IL-12 LbL-NPs promoted robust T cell accumulation in ascites and tumors in mouse models, extending survival compared to free IL-12 and remarkedly sensitizing tumors to CPI, leading to curative treatments and immune memory.