Subnanomolar Detection of Oligonucleotides through Templated Fluorogenic Reaction in Hydrogels: Controlling Diffusion to Improve Sensitivity.

Oligonucleotide-templated reactions are valuable tools for nucleic acid sensing both in vitro and in vivo. They are typically carried out under conditions that make any reaction in the absence of template highly unfavorable (most commonly by using a low concentration of reactants), which has a negative impact on the detection sensitivity. Herein, we report a novel platform for fluorogenic oligonucleotide-templated reactions between peptide nucleic acid probes embedded within permeable agarose and alginate hydrogels. We demonstrate that under conditions of restricted mobility (that is, limited diffusion), non-specific interactions between probes are prevented, thus leading to lower background signals. When applied to nucleic acid sensing, this accounts for a significant increase in sensitivity (that is, lower limit of detection). Optical nucleic acid sensors based on fluorogenic peptide nucleic acid probes embedded in permeable, physically crosslinked, alginate beads were also engineered and proved capable of detecting DNA concentrations as low as 100 pm.

Role of nitric oxide in murine conventional outflow physiology.

Elevated intraocular pressure (IOP) is the main risk factor for glaucoma. Exogenous nitric oxide (NO) decreases IOP by increasing outflow facility, but whether endogenous NO production contributes to the physiological regulation of outflow facility is unclear. Outflow facility was measured by pressure-controlled perfusion in ex vivo eyes from C57BL/6 wild-type (WT) or transgenic mice expressing human endothelial NO synthase (eNOS) fused to green fluorescent protein (GFP) superimposed on the endogenously expressed murine eNOS (eNOS-GFPtg). In WT mice, exogenous NO delivered by 100 µM S-nitroso-N-acetylpenicillamine (SNAP) increased outflow facility by 62 ± 28% (SD) relative to control eyes perfused with the inactive SNAP analog N-acetyl-d-penicillamine (NAP; n = 5, P = 0.016). In contrast, in eyes from eNOS-GFPtg mice, SNAP had no effect on outflow facility relative to NAP (-9 ± 4%, P = 0.40). In WT mice, the nonselective NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME, 10 µM) decreased outflow facility by 36 ± 13% (n = 5 each, P = 0.012), but 100 µM l-NAME had no detectable effect on outflow facility (-16 ± 5%, P = 0.22). An eNOS-selective inhibitor (cavtratin, 50 µM) decreased outflow facility by 19 ± 12% in WT (P = 0.011) and 39 ± 25% in eNOS-GFPtg (P = 0.014) mice. In the conventional outflow pathway of eNOS-GFPtg mice, eNOS-GFP expression was localized to endothelial cells lining Schlemm's canal and the downstream vessels, with no apparent expression in the trabecular meshwork. These results suggest that endogenous NO production by eNOS within endothelial cells of Schlemm's canal or downstream vessels contributes to the physiological regulation of aqueous humor outflow facility in mice, representing a viable strategy to more successfully lower IOP in glaucoma.

CLN-617 Retains IL2 and IL12 in Injected Tumors to Drive Robust and Systemic Immune-Mediated Antitumor Activity.

Despite clinical evidence of antitumor activity, the development of cytokine therapies has been hampered by a narrow therapeutic window and limited response rates. Two cytokines of high interest for clinical development are interleukin 2 (IL2) and interleukin 12 (IL12), which potently synergize to promote the activation and proliferation of T cells and NK cells. However, the only approved human IL2 therapy, Proleukin, is rarely used in the clinic due to systemic toxicities, and no IL12 product has been approved to date due to severe dose-limiting toxicities. Here, we describe CLN-617, a first-in-class therapeutic for intratumoral (IT) injection that co-delivers IL2 and IL12 on a single molecule in a safe and effective manner. CLN-617 is a single-chain fusion protein comprised of IL2, leukocyte-associated immunoglobulin-like receptor 2 (LAIR2), human serum albumin (HSA), and IL12. LAIR2 and HSA function to retain CLN-617 in the treated tumor by binding collagen and increasing molecular weight, respectively. We found that IT administration of a murine surrogate of CLN-617, mCLN-617, eradicated established treated and untreated tumors in syngeneic models, significantly improved response to anti-PD1 checkpoint therapy, and generated a robust abscopal response dependent on cellular immunity and antigen cross-presentation. CLN-617 is being evaluated in a clinical trial in patients with advanced solid tumors (NCT06035744).

Optimization of an alum-anchored clinical HIV vaccine candidate.

In the ongoing effort to develop a vaccine against HIV, vaccine approaches that promote strong germinal center (GC) responses may be critical to enable the selection and affinity maturation of rare B cell clones capable of evolving to produce broadly neutralizing antibodies. We previously demonstrated an approach for enhancing GC responses and overall humoral immunity elicited by alum-adjuvanted protein immunization via the use of phosphoserine (pSer) peptide-tagged immunogens that stably anchor to alum particles via ligand exchange with the alum particle surface. Here, using a clinically relevant stabilized HIV Env trimer termed MD39, we systematically evaluated the impact of several parameters relevant to pSer tag composition and trimer immunogen design to optimize this approach, including phosphate valency, amino acid sequence of the trimer C-terminus used for pSer tag conjugation, and structure of the pSer tag. We also tested the impact of co-administering a potent saponin/monophosphoryl lipid A (MPLA) nanoparticle co-adjuvant with alum-bound trimers. We identified MD39 trimer sequences bearing an optimized positively-charged C-terminal amino acid sequence, which, when conjugated to a pSer tag with four phosphates and a polypeptide spacer, bound very tightly to alum particles while retaining a native Env-like antigenicity profile. This optimized pSer-trimer design elicited robust antigen-specific GC B cell and serum IgG responses in mice. Through this optimization, we present a favorable MD39-pSer immunogen construct for clinical translation.

Co-Anchoring of Engineered Immunogen and Immunostimulatory Cytokines to Alum Promotes Enhanced-Humoral Immunity.

Protein antigens are often combined with aluminum hydroxide (alum), the most commonly used adjuvant in licensed vaccines; yet the immunogenicity of alum-adjuvanted vaccines leaves much room for improvement. Here, the authors demonstrate a strategy for codelivering an immunostimulatory cytokine, the interleukin IL-21, with an engineered outer domain (eOD) human immunodeficiency virus gp120 Env immunogen eOD, bound together to alum to bolster the humoral immune response. In this approach, the immunogen and cytokine are co-anchored to alum particles via a short phosphoserine (pSer) peptide linker, promoting stable binding to alum and sustained bioavailability following injection. pSer-modified eOD and IL-21 promote enhanced lymphatic drainage and lead to accumulation of the vaccine in B cell follicles in the draining lymph nodes. This in turn promotes enhanced T follicular helper cell priming and robust germinal center responses as well as increased antigen-specific serum IgG titers. This is a general strategy for codelivery of immunostimulatory cytokine with immunogens providing a facile approach to modulate T cell priming and GC reactions toward enhanced protective immunity using the most common clinical vaccine adjuvant.

Intratumourally injected alum-tethered cytokines elicit potent and safer local and systemic anticancer immunity.

Anti-tumour inflammatory cytokines are highly toxic when administered systemically. Here, in multiple syngeneic mouse models, we show that the intratumoural injection of recombinantly expressed cytokines bound tightly to the common vaccine adjuvant aluminium hydroxide (alum) (via ligand exchange between hydroxyls on the surface of alum and phosphoserine residues tagged to the cytokine by an alum-binding peptide) leads to weeks-long retention of the cytokines in the tumours, with minimal side effects. Specifically, a single dose of alum-tethered interleukin-12 induced substantial interferon-γ-mediated T-cell and natural-killer-cell activities in murine melanoma tumours, increased tumour antigen accumulation in draining lymph nodes and elicited robust tumour-specific T-cell priming. Moreover, intratumoural injection of alum-anchored cytokines enhanced responses to checkpoint blockade, promoting cures in distinct poorly immunogenic syngeneic tumour models and eliciting control over metastases and distant untreated lesions. Intratumoural treatment with alum-anchored cytokines represents a safer and tumour-agnostic strategy to improving local and systemic anticancer immunity.

Intranasal vaccination with lipid-conjugated immunogens promotes antigen transmucosal uptake to drive mucosal and systemic immunity.

To combat the HIV epidemic and emerging threats such as SARS-CoV-2, immunization strategies are needed that elicit protection at mucosal portals of pathogen entry. Immunization directly through airway surfaces is effective in driving mucosal immunity, but poor vaccine uptake across the mucus and epithelial lining is a limitation. The major blood protein albumin is constitutively transcytosed bidirectionally across the airway epithelium through interactions with neonatal Fc receptors (FcRn). Exploiting this biology, here, we demonstrate a strategy of "albumin hitchhiking" to promote mucosal immunity using an intranasal vaccine consisting of protein immunogens modified with an amphiphilic albumin-binding polymer-lipid tail, forming amph-proteins. Amph-proteins persisted in the nasal mucosa of mice and nonhuman primates and exhibited increased uptake into the tissue in an FcRn-dependent manner, leading to enhanced germinal center responses in nasal-associated lymphoid tissue. Intranasal immunization with amph-conjugated HIV Env gp120 or SARS-CoV-2 receptor binding domain (RBD) proteins elicited 100- to 1000-fold higher antigen-specific IgG and IgA titers in the serum, upper and lower respiratory mucosa, and distal genitourinary mucosae of mice compared to unmodified protein. Amph-RBD immunization induced high titers of SARS-CoV-2-neutralizing antibodies in serum, nasal washes, and bronchoalveolar lavage. Furthermore, intranasal amph-protein immunization in rhesus macaques elicited 10-fold higher antigen-specific IgG and IgA responses in the serum and nasal mucosa compared to unmodified protein, supporting the translational potential of this approach. These results suggest that using amph-protein vaccines to deliver antigen across mucosal epithelia is a promising strategy to promote mucosal immunity against HIV, SARS-CoV-2, and other infectious diseases.

Catalytic antibody degradation of ghrelin increases whole-body metabolic rate and reduces refeeding in fasting mice.

Obesity is a chronic, costly, and globally prevalent condition, with excess caloric intake a suspected etiologic factor. Nonsurgical treatments are modestly efficacious, and weight loss maintenance is hampered by anti-famine homeostatic mechanisms. Ghrelin, a gastric hormone linked to meal initiation, energy expenditure, and fuel partitioning, is hypothesized to facilitate weight gain and impede weight loss. Unique among known animal peptides, the serine-3 residue of ghrelin is posttranslationally acylated with an n-octanoic acid, a modification important for the peptide's active blood-brain transport and growth hormone secretagogue receptor-1 agonist activity. Pharmacological degradation of ghrelin would be hypothesized to reduce ghrelin's biological effects. To study endogenous ghrelin's role in appetite and energy expenditure, we generated antibodies that hydrolyze the octanoyl moiety of ghrelin to form des-acyl ghrelin. The most proficient antibody catalyst, GHR-11E11, was found to display a second-order rate constant of 18 M(-1) x s(-1) for the hydrolysis of ghrelin to des-acyl ghrelin. I.v. administration of GHR-11E11 (50 mg/kg) maintained a greater metabolic rate in fasting C57BL/6J mice as compared with mice receiving a control antibody and suppressed 6-h refeeding after 24 h of food deprivation. Indirect respiratory measures of metabolism after refeeding and relative fuel substrate utilization were unaffected. The results support the hypothesis that acylated ghrelin stimulates appetite and curbs energy expenditure during deficient energy intake, whereas des-acyl ghrelin does not potently share these functions. Catalytic anti-ghrelin antibodies might thereby adjunctively aid consolidation of caloric restriction-induced weight loss and might also be therapeutically relevant to Prader-Willi syndrome, characterized after infancy by hyperghrelinemia, hyperphagia, and obesity.

The effect of tai chi on cognition in elders with cognitive impairment.

This one-arm pilot study investigated the effect of tai chi on cognition in elders with cognitive impairment. Although no significant difference existed between pre- and post-test performance on all cognition measures, a dose-response relationship was demonstrated between attendance and some cognition measures.

Phosphate-mediated coanchoring of RBD immunogens and molecular adjuvants to alum potentiates humoral immunity against SARS-CoV-2.

There is a need for additional rapidly scalable, low-cost vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to achieve global vaccination. Aluminum hydroxide (alum) adjuvant is the most widely available vaccine adjuvant but elicits modest humoral responses. We hypothesized that phosphate-mediated coanchoring of the receptor binding domain (RBD) of SARS-CoV-2 together with molecular adjuvants on alum particles could potentiate humoral immunity by promoting extended vaccine kinetics and codelivery of vaccine components to lymph nodes. Modification of RBD immunogens with phosphoserine (pSer) peptides enabled efficient alum binding and slowed antigen clearance, leading to notable increases in germinal center responses and neutralizing antibody titers in mice. Adding phosphate-containing CpG or saponin adjuvants to pSer-RBD:alum immunizations synergistically enhanced vaccine immunogenicity in mice and rhesus macaques, inducing neutralizing responses against SARS-CoV-2 variants. Thus, phosphate-mediated coanchoring of RBD and molecular adjuvants to alum is an effective strategy to enhance the efficacy of SARS-CoV-2 subunit vaccines.

A particulate saponin/TLR agonist vaccine adjuvant alters lymph flow and modulates adaptive immunity.

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 immune-stimulatory complexes (ISCOMs) formed by the self-assembly of saponin and phospholipids in the absence or presence of the Toll-like receptor 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 cell­dependent manner and promoting antigen entry into draining lymph nodes. SMNP was particularly effective, exhibiting even greater potency than the compositionally related adjuvant AS01B in mice, and primed robust germinal center B cell, TFH, and HIV tier 2 neutralizing antibodies in nonhuman primates. Together, these findings shed new light on mechanisms by which saponin adjuvants act to promote the immune response and suggest that SMNP may be a promising adjuvant in the setting of HIV, SARS-CoV-2, and other pathogens.

Engineered immunogen binding to alum adjuvant enhances humoral immunity.

Adjuvants are central to the efficacy of subunit vaccines. Aluminum hydroxide (alum) is the most commonly used vaccine adjuvant, yet its adjuvanticity is often weak and mechanisms of triggering antibody responses remain poorly understood. We demonstrate that site-specific modification of immunogens with short peptides composed of repeating phosphoserine (pSer) residues enhances binding to alum and prolongs immunogen bioavailability. The pSer-modified immunogens formulated in alum elicited greatly increased germinal center, antibody, neutralizing antibody, memory and long-lived plasma cell responses compared to conventional alum-adsorbed immunogens. Mechanistically, pSer-immunogen:alum complexes form nanoparticles that traffic to lymph nodes and trigger B cell activation through multivalent and oriented antigen display. Direct uptake of antigen-decorated alum particles by B cells upregulated antigen processing and presentation pathways, further enhancing B cell activation. These data provide insights into mechanisms of action of alum and introduce a readily translatable approach to significantly improve humoral immunity to subunit vaccines using a clinical adjuvant.

Author Correction: A Engineered immunogen binding to alum adjuvant enhances humoral immunity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The effect of tai chi on knee osteoarthritis pain in cognitively impaired elders: pilot study.

This article reports a pilot study of the effect of tai chi (TC), a pharmacological adjunct and mild aerobic exercise, on osteoarthritic knee pain in elders with cognitive impairment (CI). The TC program included a warm-up, 12-form Sun-style TC, and a cool-down period, for a total of 20-40 minutes per session, twice a week for 15 weeks. The results showed no significant differences in knee pain after the TC intervention in 7 elders with CI. However, more minutes of TC attendance were related to improved pain scores (Spearman's rho=.78, P < .05). Greater accuracy in TC performance was also correlated with improvements in pain scores (Spearman's rho = .70, P=.08). Of 4 elders who participated in TC practice regularly (more than 20 sessions), 3 showed clinically important improvements, but 3 elders who participated in no sessions or only a few sessions showed no improvement.

Hydrogel-Coated Microneedle Arrays for Minimally Invasive Sampling and Sensing of Specific Circulating Nucleic Acids from Skin Interstitial Fluid.

Minimally invasive technologies that can sample and detect cell-free nucleic acid biomarkers from liquid biopsies have recently emerged as clinically useful for early diagnosis of a broad range of pathologies, including cancer. Although blood has so far been the most commonly interrogated bodily fluid, skin interstitial fluid has been mostly overlooked despite containing the same broad variety of molecular biomarkers originating from cells and surrounding blood capillaries. Emerging technologies to sample this fluid in a pain-free and minimally-invasive manner often take the form of microneedle patches. Herein, we developed microneedles that are coated with an alginate-peptide nucleic acid hybrid material for sequence-specific sampling, isolation, and detection of nucleic acid biomarkers from skin interstitial fluid. Characterized by fast sampling kinetics and large sampling capacity (∼6.5 µL in 2 min), this platform technology also enables the detection of specific nucleic acid biomarkers either on the patch itself or in solution after light-triggered release from the hydrogel. Considering the emergence of cell-free nucleic acids in bodily fluids as clinically informative biomarkers, platform technologies that can detect them in an automated and minimally invasive fashion have great potential for personalized diagnosis and longitudinal monitoring of patient-specific disease progression.

Correction: Oligonucleotide-templated lateral flow assays for amplification-free sensing of circulating microRNAs.

Correction for 'Oligonucleotide-templated lateral flow assays for amplification-free sensing of circulating microRNAs' by Suraj Pavagada et al., Chem. Commun., 2019, 55, 12451-12454.

Oligonucleotide-templated lateral flow assays for amplification-free sensing of circulating microRNAs.

Herein we demonstrate the first example of oligonucleotide-templated reaction (OTR) performed on paper, using lateral flow to capture and concentrate specific nucleic acid biomarkers on a test line. Quantitative analysis, using a low-cost benchtop fluorescence reader showed very high specificity down to the single nucleotide level and proved sensitive enough for amplification-free, on-chip, detection of endogenous concentrations of miR-150-5p, a recently identified predictive blood biomarker for preterm birth.

Enhanced CAR-T cell activity against solid tumors by vaccine boosting through the chimeric receptor.

Chimeric antigen receptor-T cell (CAR-T) therapy has been effective in the treatment of hematologic malignancies, but it has shown limited efficacy against solid tumors. Here we demonstrate an approach to enhancing CAR-T function in solid tumors by directly vaccine-boosting donor cells through their chimeric receptor in vivo. We designed amphiphile CAR-T ligands (amph-ligands) that, upon injection, trafficked to lymph nodes and decorated the surfaces of antigen-presenting cells, thereby priming CAR-Ts in the native lymph node microenvironment. Amph-ligand boosting triggered massive CAR-T expansion, increased donor cell polyfunctionality, and enhanced antitumor efficacy in multiple immunocompetent mouse tumor models. We demonstrate two approaches to generalizing this strategy to any chimeric antigen receptor, enabling this simple non-human leukocyte antigen-restricted approach to enhanced CAR-T functionality to be applied to existing CAR-T designs.