Mucosal Adenoviral-vectored Vaccine Boosting Durably Prevents XBB.1.16 Infection in Nonhuman Primates.

Waning immunity and continued virus evolution have limited the durability of protection from symptomatic infection mediated by intramuscularly (IM)-delivered mRNA vaccines against COVID-19 although protection from severe disease remains high. Mucosal vaccination has been proposed as a strategy to increase protection at the site of SARS-CoV-2 infection by enhancing airway immunity, potentially reducing rates of infection and transmission. Here, we compared protection against XBB.1.16 virus challenge 5 months following IM or mucosal boosting in non-human primates (NHP) that had previously received a two-dose mRNA-1273 primary vaccine regimen. The mucosal boost was composed of a bivalent chimpanzee adenoviral-vectored vaccine encoding for both SARS-CoV-2 WA1 and BA.5 spike proteins (ChAd-SARS-CoV-2-S) and delivered either by an intranasal mist or an inhaled aerosol. An additional group of animals was boosted by the IM route with bivalent WA1/BA.5 spike-matched mRNA (mRNA-1273.222) as a benchmark control. NHP were challenged in the upper and lower airways 18 weeks after boosting with XBB.1.16, a heterologous Omicron lineage strain. Cohorts boosted with ChAd-SARS-CoV-2-S by an aerosolized or intranasal route had low to undetectable virus replication as assessed by levels of subgenomic SARS-CoV-2 RNA in the lungs and nose, respectively. In contrast, animals that received the mRNA-1273.222 boost by the IM route showed minimal protection against virus replication in the upper airway but substantial reduction of virus RNA levels in the lower airway. Immune analysis showed that the mucosal vaccines elicited more durable antibody and T cell responses than the IM vaccine. Protection elicited by the aerosolized vaccine was associated with mucosal IgG and IgA responses, whereas protection elicited by intranasal delivery was mediated primarily by mucosal IgA. Thus, durable immunity and effective protection against a highly transmissible heterologous variant in both the upper and lower airways can be achieved by mucosal delivery of a virus-vectored vaccine. Our study provides a template for the development of mucosal vaccines that limit infection and transmission against respiratory pathogens.

Divergence of variant binding/neutralizing antibodies following SARS-CoV-2 booster vaccines in myeloma: Impact of hybrid immunity.

We characterized virus-neutralization and spike-binding antibody profiles in myeloma patients following monovalent or bivalent-SARS-CoV-2 booster vaccination. Vaccination improves the breadth of binding antibodies but not neutralization activity against current variants. Hybrid immunity and immune imprinting impact vaccine-elicited immunity.

Divergence of variant binding/neutralizing antibodies following SARS-CoV-2 booster vaccines in myeloma: Impact of hybrid immunity.

We characterized virus-neutralization and spike-binding antibody profiles in myeloma patients following monovalent or bivalent-SARS-CoV-2 booster vaccination. Vaccination improves the breadth of binding antibodies but not neutralization activity against current variants. Hybrid immunity and immune imprinting impact vaccine-elicited immunity.

Impaired SARS-CoV-2 Variant Neutralization and CD8+ T-cell Responses Following 3 Doses of mRNA Vaccines in Myeloma: Correlation with Breakthrough Infections.

Patients with multiple myeloma (MM) mount suboptimal neutralizing antibodies (nAb) following 2 doses of SARS-CoV-2 mRNA vaccines. Currently, circulating SARS-CoV-2 variants of concern (VOC) carry the risk of breakthrough infections. We evaluated immune recognition of current VOC including BA.1, BA.2, and BA.5 in 331 racially representative patients with MM following 2 or 3 doses of mRNA vaccines. The third dose increased nAbs against WA1 in 82%, but against BA variants in only 33% to 44% of patients. Vaccine-induced nAbs correlated with receptor-binding domain (RBD)-specific class-switched memory B cells. Vaccine-induced spike-specific T cells were detected in patients without seroconversion and cross-recognized variant-specific peptides but were predominantly CD4+ T cells. Detailed clinical/immunophenotypic analysis identified features correlating with nAb/B/T-cell responses. Patients who developed breakthrough infections following 3 vaccine doses had lower live-virus nAbs, including against VOC. Patients with MM remain susceptible to SARS-CoV-2 variants following 3 vaccine doses and should be prioritized for emerging approaches to elicit variant-nAb and CD8+ T cells. SIGNIFICANCE: Three doses of SARS-CoV-2 mRNA vaccines fail to yield detectable VOC nAbs in nearly 60% and spike-specific CD8+ T cells in >80% of myeloma patients. Patients who develop breakthrough infections following vaccination have low levels of live-virus nAb. This article is highlighted in the In This Issue feature, p. 101.

Osteopontin as a biomarker for COVID-19 severity and multisystem inflammatory syndrome in children: A pilot study.

This study sought to evaluate the candidacy of plasma osteopontin (OPN) as a biomarker of COVID-19 severity and multisystem inflammatory condition in children (MIS-C) in children. A retrospective analysis of 26 children (0-21 years of age) admitted to Children's Healthcare of Atlanta with a diagnosis of COVID-19 between March 17 and May 26, 2020 was undertaken. The patients were classified into three categories based on COVID-19 severity levels: asymptomatic or minimally symptomatic (control population, admitted for other non-COVID-19 conditions), mild/moderate, and severe COVID-19. A fourth category of children met the Centers for Disease Control and Prevention's case definition for MIS-C. Residual blood samples were analyzed for OPN, a marker of inflammation using commercial ELISA kits (R&D), and results were correlated with clinical data. This study demonstrates that OPN levels are significantly elevated in children hospitalized with moderate and severe COVID-19 and MIS-C compared to OPN levels in mild/asymptomatic children. Further, OPN differentiated among clinical levels of severity in COVID-19, while other inflammatory markers including maximum erythrocyte sedimentation rate, C-reactive protein and ferritin, minimum lymphocyte and platelet counts, soluble interleukin-2R, and interleukin-6 did not. We conclude OPN is a potential biomarker of COVID-19 severity and MIS-C in children that may have future clinical utility. The specificity and positive predictive value of this marker for COVID-19 and MIS-C are areas for future larger prospective research studies.

Prophylactic progesterone prevents adverse behavioural and neurocognitive effects of neonatal anaesthesia exposure in rat.

BACKGROUND: Evidence from animal models and human studies suggests an association between early general anaesthesia exposure and development of long-lasting neurocognitive problems including learning and memory impairments and an anxious phenotype. Because millions of children each year undergo procedures that require anaesthesia, it is important to investigate ways to protect the vulnerable developing brain. We evaluated whether progesterone treatment administered before general anaesthesia exposure could prevent long-term anaesthesia-induced neurocognitive and behavioural changes. METHODS: Female and male Long-Evans rat pups were repeatedly exposed to 2 h of sevoflurane or control procedures at postnatal days 7, 10, and 13. Subcutaneous injections of progesterone or vehicle were administered immediately before general anaesthesia exposure or control procedures. Neurobehavioural and cognitive outcomes were evaluated using elevated plus maze and Morris water maze tests. RESULTS: Prophylactic progesterone treatment attenuated the chemokine (C-X-C motif) ligand 1 (CXCL1) response to sevoflurane exposure. Rats given vehicle treatment with general anaesthesia exposure exhibited increased anxiety on the elevated plus maze and learning and memory impairments on the Morris water maze. However, rats treated with progesterone before general anaesthesia lacked these impairments and performed in a similar manner to controls on both tasks. CONCLUSIONS: Progesterone attenuated the anaesthesia-induced, acute peripheral inflammatory response and prevented cognitive and behavioural alterations associated with early repeated general anaesthesia exposure. Importantly, our results suggest that progesterone treatments given before general anaesthesia may help to protect the developing brain.

Neurocognitive Outcomes in a Cisternal Blood Injection Murine Model of Subarachnoid Hemorrhage.

BACKGROUND: Subarachnoid hemorrhage (SAH) results in neurocognitive dysfunction and anxiety in humans and in animal models. Neurobehavioral tests such as the Morris Water Maze (MWM) and Elevated Plus Maze (EPM) tests are validated in several models of SAH but have not been tested in the murine cisternal blood injection SAH model. METHODS: Adult C57BL/6 mice (n=16) were randomized into two groups. Group 1 (n=8) received sham surgery. Group 2 (n=8) underwent SAH with 60 µL of autologous blood injected into the cisterna magna. Mice were then tested using the Modified Garcia Score on post-operative day 2 (POD2), EPM on POD5 & POD16, and MWM on POD6-16.Brain tissues harvested on POD16 were stained with Fluoro-Jade C to identify neurodegeneration in the hippocampus and cortex and Iba-1 immunofluorescence staining for microglial activation in the dentate gyrus and CA1 region of the hippocampus. RESULTS: SAH mice showed increased escape latency on POD10. Swim distance was significantly increased on POD9-10 and swim speed was significantly decreased on POD6&POD10 in SAH mice. SAH mice exhibited a trend for lowered proportion of covered arm entries in EPM on POD16. Modified Garcia Score was not significantly different between the groups on POD2. The area of microglial activation in the dentate gyrus and CA1 region of the hippocampus was mildly increased but not significantly different at day 16 after SAH. Similarly, no significant differences were noted in the number of Fluoro-Jade C (+) cells in cortex or hippocampus. CONCLUSIONS: Cisternal single blood injection in mice produces mild neurocognitive deficits most pronounced in spatial learning and most evident 10 days after SAH.

Development of a novel progesterone analog in the treatment of traumatic brain injury.

Although systemic progesterone (PROG) treatment has been shown to be neuroprotective by many laboratories and in multiple animal models of brain injury including traumatic brain injury (TBI), PROG's poor aqueous solubility limits its potential for use as a therapeutic agent. The problem of solubility presents challenges for an acute intervention for neural injury, when getting a neuroprotectant to the brain quickly is crucial. Native PROG (nPROG) is hydrophobic and does not readily dissolve in an aqueous-based medium, so this makes it harder to give under emergency field conditions. An agent with properties similar to those of PROG but easier to store, transport, formulate, and administer early in emergency trauma situations could lead to better and more consistent clinical outcomes following TBI. At the same time, the engineering of a new molecule designed to treat a complex systemic injury must anticipate a range of translational issues including solubility and bioavailability. Here we describe the development of EIDD-1723, a novel, highly stable PROG analog with >104-fold higher aqueous solubility than that of nPROG. We think that, with further testing, EIDD-1723 could become an attractive candidate use as a field-ready treatment for TBI patients. This article is part of the Special Issue entitled "Novel Treatments for Traumatic Brain Injury".

Vitamin D deficiency increases blood-brain barrier dysfunction after ischemic stroke in male rats.

Because vitamin D hormone deficiency (VDHdef) can worsen severity and outcome for ischemic stroke, we examined the role of VDH in maintaining blood-brain-barrier (BBB integrity) in a rat model of stroke. In most types of stroke, the BBB is markedly compromised, potentially leading to a cascade of injury processes and functional deficits, so we examined a number of biomarkers associated with BBB disruption to determine whether VDH deficiency would further compromise the BBB following a stroke. Male Wistar rats were randomly assigned to one of two diet cohorts, VDH-sufficient (VDHsuf) and VDHdef. The VDHsuf group was fed standard rat chow and the VDHdef group got a VDH-null version of the same diet for 8 weeks. Animals from both cohorts were subjected to transient middle cerebral artery occlusion (tMCAO) surgery, killed at 72 h post-stroke, and their brains evaluated for BBB permeability and injury severity using expression of immunoglobulin (IgG), matrix metalloproteinase-9 (MMP-9) activity and alteration of tight junction (TJ) proteins as markers of BBB disruption. We also evaluated modulation of glucose transporter-1 (GLUT1), osteopontin (OPN), ß-catenin and vitamin D receptor (VDR) expression in VDHsuf and VDHdef subjects. At the time of MCAO, rats on the VDHdef diet had circulating VDH levels one-fourth that of rats fed control chow. IgG extravasation after MCAO, indicating more severe BBB injury, was significantly higher in the MCAO+VDHdef than the MCAO+VDHsuf rats. Following MCAO, expression of MMP-9, GLUT1, VDR and OPN increased and the TJ proteins occludin and claudin-5 decreased significantly in the VDHdef compared to the VDHsuf group. We also observed significantly lower expression of ß-catenin in the MCAO group of both VDHsuf and VDHdef rats. Under these conditions, VDH deficiency itself can compromise the BBB. We think that low serum VDH levels are likely to complicate stroke severity and its chronic consequences.

Effect of Progesterone on Cerebral Vasospasm and Neurobehavioral Outcomes in a Rodent Model of Subarachnoid Hemorrhage.

BACKGROUND: Subarachnoid hemorrhage (SAH) induces widespread inflammation leading to cellular injury, vasospasm, and ischemia. Evidence suggests that progesterone (PROG) can improve functional recovery in acute brain injury owing to its anti-inflammatory and neuroprotective properties, which could also be beneficial in SAH. We hypothesized that PROG treatment attenuates inflammation-mediated cerebral vasospasm and microglial activation, improves synaptic connectivity, and ameliorates functional recovery after SAH. METHODS: We investigated the effect of PROG in a cisternal SAH model in adult male C57BL/6 mice. Neurobehavioral outcomes were evaluated using rotarod latency and grip strength tests. Basilar artery perimeter, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor 1 (GluR1)/synaptophysin colocalization, and Iba-1 immunoreactivity were quantified histologically. RESULTS: PROG (8 mg/kg) significantly improved rotarod latency at day 6 and grip strength at day 9. PROG-treated mice had significantly reduced basilar artery vasospasm at 24 hours. GluR1/synaptophysin colocalization, indicative of synaptic GluR1, was significantly reduced in the SAH+Vehicle group at 24 hours, and PROG treatment significantly attenuated this reduction. PROG treatment significantly reduced microglial cell activation and proliferation in cerebellum and cortex but not in the brainstem at 10 days. CONCLUSIONS: PROG treatment ameliorated cerebral vasospasm, reduced microglial activation, restored synaptic GluR1 localization, and improved neurobehavioral performance in a murine model of SAH. These results provide a rationale for further translational testing of PROG therapy in SAH.

Intralipid Vehicle Does Not Interfere with the Efficacy of Progesterone in Attenuating Edema following Traumatic Brain Injury.

The recent disappointing results of phase III trials for progesterone (PROG) in traumatic brain injury (TBI) have triggered speculation about reasons for the negative outcomes. One confounding factor may have been the vehicle used to administer PROG. Virtually all of the many pre-clinical experiments informing the clinical trials and reporting beneficial PROG effects used more soluble 2-hydroxypropyl-b-cyclodextrin as a vehicle given intraperitoneally or subcutaneously rather than a lipid formulation given intravenously (IV). The present investigation compared the effect of PROG infusion with that of lipid emulsion (Intralipid®) as a carrier/vehicle on edema following TBI in rats. Eight-mg/kg doses of PROG with 20% Intralipid were given IV via central venous catheter beginning 1 h post-injury over a 1 h duration (1.2 mL/h). Animals were killed and brains removed at 24 h post-injury. All the brain-injured groups showed more edema compared with the control group. However, PROG+Intralipid significantly attenuated cerebral swelling compared with Intralipid alone. No difference was observed between the TBI-alone and Intralipid groups. Although this study used much a smaller volume and shorter duration of Intralipid infusion than the clinical trials (up to 5 days of continuous infusion), our results suggest that the use of Intralipid in rats did not prevent or mask the beneficial effect of PROG.

Evaluating the neurotherapeutic potential of a water-soluble progesterone analog after traumatic brain injury in rats.

The poor aqueous solubility of progesterone (PROG) limits its potential use as a therapeutic agent. We designed and tested EIDD-1723, a novel water-soluble analog of PROG with >100-fold higher solubility than that of native PROG, as candidate for development as a field-ready treatment for traumatic brain injury (TBI). The pharmacokinetic effects of EIDD-1723 on morphological and functional outcomes in rats with bilateral cortical impact injury were evaluated. Following TBI, 10-mg/kg doses of EIDD-1723 or PROG were given intramuscularly (i.m.) at 1, 6 and 24 h post-injury, then daily for the next 6 days, with tapering of the last 2 treatments. Rats were tested pre-injury to establish baseline performance on grip strength and sensory neglect, and then retested at 4, 9 and 21 days post-TBI. Spatial learning was evaluated from days 11-17 post-TBI. At 22 days post-injury, rats were perfused and brains extracted and processed for lesion size. For the edema assay the animals were killed and brains removed at 24 h post-injury. EIDD-1723 significantly reduced cerebral edema and improved recovery from motor, sensory and spatial learning deficits as well as, or better than, native PROG. Pharmacokinetic investigation after a single i.m. injection in rats revealed that EIDD-1723 was rapidly converted to the active metabolite EIDD-036, demonstrating first-order elimination kinetics and ability to cross the blood-brain barrier. Our results suggest that EIDD-1723 represents a substantial advantage over current PROG formulations because it overcomes storage, formulation and delivery limitations of PROG and can thereby reduce the time between injury and treatment.

Progesterone improves long-term functional and histological outcomes after permanent stroke in older rats.

Previous studies have shown progesterone to be beneficial in animal models of central nervous system injury, but less is known about its longer-term sustained effects on recovery of function following stroke. We evaluated progesterone's effects on a panel of behavioral tests up to 8 weeks after permanent middle cerebral artery occlusion (pMCAO). Male Sprague-Dawley rats 12m.o. were subjected to pMCAO and, beginning 3h post-pMCAO, given intraperitoneal injections of progesterone (8mg/kg) or vehicle, followed by subcutaneous injections at 8h and then every 24h for 7 days, with tapering of the last 2 treatments. The rats were then tested on functional recovery at 3, 6 and 8 weeks post-stroke. We observed that progesterone-treated animals showed attenuation of infarct volume and improved functional outcomes at 8 weeks after stroke on grip strength, sensory neglect, motor coordination and spatial navigation tests. Progesterone treatments significantly improved motor deficits in the affected limb on a number of gait parameters. Glial fibrillary acidic protein expression was increased in the vehicle group and considerably lowered in the progesterone group at 8 weeks post-stroke. With repeated post-stroke testing, sensory neglect and some aspects of spatial learning performance showed spontaneous recovery, but on gait and grip-strength measres progesterone given only in the acute stage of stroke (first 7 days) showed sustained beneficial effects on all other measures of functional recovery up to 8 weeks post-stroke.

Progesterone in experimental permanent stroke: a dose-response and therapeutic time-window study.

Currently, the only approved treatment for ischaemic stroke is tissue plasminogen activator, a clot-buster. This treatment can have dangerous consequences if not given within the first 4 h after stroke. Our group and others have shown progesterone to be beneficial in preclinical studies of stroke, but a progesterone dose-response and time-window study is lacking. We tested male Sprague-Dawley rats (12 months old) with permanent middle cerebral artery occlusion or sham operations on multiple measures of sensory, motor and cognitive performance. For the dose-response study, animals received intraperitoneal injections of progesterone (8, 16 or 32 mg/kg) at 1 h post-occlusion, and subcutaneous injections at 6 h and then once every 24 h for 7 days. For the time-window study, the optimal dose of progesterone was given starting at 3, 6 or 24 h post-stroke. Behavioural recovery was evaluated at repeated intervals. Rats were killed at 22 days post-stroke and brains extracted for evaluation of infarct volume. Both 8 and 16 mg/kg doses of progesterone produced attenuation of infarct volume compared with the placebo, and improved functional outcomes up to 3 weeks after stroke on locomotor activity, grip strength, sensory neglect, gait impairment, motor coordination and spatial navigation tests. In the time-window study, the progesterone group exhibited substantial neuroprotection as late as 6 h after stroke onset. Compared with placebo, progesterone showed a significant reduction in infarct size with 3- and 6-h delays. Moderate doses (8 and 16 mg/kg) of progesterone reduced infarct size and improved functional deficits in our clinically relevant model of stroke. The 8 mg/kg dose was optimal in improving motor, sensory and memory function, and this effect was observed over a large therapeutic time window. Progesterone shows promise as a potential therapeutic agent and should be examined for safety and efficacy in a clinical trial for ischaemic stroke.

Progesterone attenuates hemorrhagic transformation after delayed tPA treatment in an experimental model of stroke in rats: involvement of the VEGF-MMP pathway.

Tissue plasminogen activator (tPA) is the only FDA-approved treatment for acute stroke, but its use remains limited. Progesterone (PROG) has shown neuroprotection in ischemia, but before clinical testing, we must determine how it affects hemorrhagic transformation in tPA-treated ischemic rats. Male Sprague-Dawley rats underwent middle cerebral artery occlusion with reperfusion at 4.5 hours and tPA treatment at 4.5 hours, or PROG treatment intraperitoneally at 2 hours followed by subcutaneous injection at 6 hours post occlusion. Rats were killed at 24 hours and brains evaluated for cerebral hemorrhage, swelling, blood-brain barrier (BBB) permeability, and levels of matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor level (VEGF), and tight junction (TJ) proteins. We also evaluated PROG's efficacy in preventing tPA-induced impairment of transendothelial electrical resistance (TEER) and TJ proteins under hypoxia/reoxygenation in the endothelial cells. Delayed tPA treatment induced significant hemorrhagic conversion and brain swelling. Treatment with PROG plus tPA ameliorated hemorrhage, hemispheric swelling, BBB permeability, MMP-9 induction, and VEGF levels compared with controls. Progesterone treatment significantly prevented tPA-induced decrease in TEER and expression of occludin and claudin-5, and attenuated VEGF levels in culture media subjected to hypoxia. The study concluded that PROG may extend the time window for tPA administration in ischemic stroke and reduce hemorrhagic conversion.

Improved behavioral outcomes after progesterone administration in aged male rats with traumatic brain injury.

Twenty-month-old male Fischer 344 rats with bilateral contusions of the frontal cortex (n=8/group) or sham operations received 16 mg/kg of progesterone or vehicle at 1 and 6 h post-injury, then once every 24 h for the next seven days, with tapering of the dose over the final two treatments. The rats' behavioral recovery was then evaluated on tests of locomotor activity and Morris water maze learning. All rats were sacrificed 21 days post-TBI and brains were perfused and cryoprotected for necrotic cavity measurements. The injury produced significant impairments in activity and spatial learning compared to sham controls. The progesterone-treated rats had better functional outcomes than vehicle-treated rats with similar cortical injuries. The neurosteroid treatment did not affect the size of the necrotic cavity.

Direct inhibition of the mitochondrial permeability transition pore: a possible mechanism for better neuroprotective effects of allopregnanolone over progesterone.

We previously demonstrated that the progesterone (PROG) metabolite allopregnanolone (AP) is more potent than PROG in the treatment of traumatic brain injury (TBI) and stroke, but the mechanisms for this differential effect are little understood. The mitochondrial permeability transition pore (mtPTP) appears to be a key player in the intrinsic pathway of apoptosis-induced loss of neurons. Its activation is accompanied by the release of cytochrome c (cyt c) from the intermembrane gap and subsequent cell death. We investigated whether mtPTP is implicated in the mechanisms of PROG and AP neuroprotection following traumatic and ischemic brain injury. To assess the neurosteroids' direct effects on mtPTP activity at the single-channel level, recordings from the inner mitochondrial membrane were obtained by a patch-clamp approach in rat liver mitoplasts. AP but not PROG strongly inhibited mtPTP currents. Interaction of AP with the PTP was further supported by a swelling assay demonstrating that AP inhibited Ca(2+)-triggered swelling in functionally intact rat liver and brain mitochondria. If AP inhibits the mtPTP, it should prevent the mitochondrial cyt c release seen in stroke and TBI. To test this idea, we subjected one group of rats to cortical contusion injury (CCI) and another to transient middle cerebral artery occlusion (MCAO). AP-treated animals showed substantially decreased cyt c release and AP was more potent than PROG in inhibiting mitochondrial cyt c release at 24 h post-CCI and -MCAO. Our results demonstrate that AP inhibits the mtPTP current. This may help to explain its more potent anti-apoptotic and neuroprotective effects compared to PROG.