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A STING (stimulator of interferon genes) agonist GSK3996915 under investigation in early discovery for hepatitis B was orally dosed to a mouse model for understanding the parent drug distribution in liver, the target organ. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was used to quantify the distribution of GSK3996915 in liver collected from mice administered a single oral dose at 90 mg/kg. GSK3996915 was detected with a zonal distribution localized in the portal triad and highly concentrated in the main bile ducts, indicating clearance through biliary excretion. High spatial resolution imaging showed the distribution of the parent drug localized to the cellular populations in the sinusoids, including the Kupffer cells. Additionally, a series of drug-related metabolites were observed to be localized in the central zones of the liver. These results exemplify the potential of utilizing MALDI IMS for measuring not only quantitative drug distribution and target exposure but also drug metabolism and elimination in a single suite of experiments. SIGNIFICANCE STATEMENT: An integrated imaging approach utilizing matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) complemented with immunohistochemistry (IHC) and histology was used to address the question of target exposure at the cellular level. Localized quantification of the parent drug in the target organ and identification of potential metabolites in the context of tissue histology were also achieved in one experimental suite to support characterization of pharmacokinetic properties of the drug in the early discovery stage., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Vaccines containing mRNA with the capacity to self-amplify represent an alternative to the mRNA vaccines that came to prominence during the COVID-19 pandemic. To gain further insights on the safety profile of self-amplifying mRNA- (SAM-) vaccines, this preclinical toxicology study in rats evaluated the effect of (i) the type of delivery system (lipid nanoparticle [LNP] vs cationic nano-emulsion [CNE]); (ii) antigen-encoding sequence (rabies glycoprotein G vs SARS-CoV-2 Spike); and (iii) RNA amplification. Further analyses also evaluated gene expression in peripheral blood after vaccination, and the biodistribution of vaccine RNA. The SAM vaccines administered as two doses 2-weeks apart had acceptable safety profiles in rats, with respect to clinical signs, blood biochemistry, and macroscopic and microscopic pathology. A transient increase in ALT/AST ratio occurred only in female rats and in the absence of muscle and liver damage was dependent on RNA amplification and appeared related to the greater quantities of vaccine RNA in the muscle and livers of female rats vs male rats. The RNA and delivery-vehicle components, but not the nature of the antigen-coding sequence or the requirement for RNA amplification, affected aspects of the stimulation of innate-immune activity, which was consistent with the transient activation of type I and type II interferon signaling. The delivery vehicle, LNP, differed from CNE as vaccine RNA in CNE compositions appeared independently to stimulate innate-immune activity at 4 hours after vaccination. Our analysis supports further studies to assess whether these differences in innate-immune activity affect safety and efficacy of the SAM vaccine.

RNA vaccines have demonstrated efficacy against SARS-CoV-2 in humans, and the technology is being leveraged for rapid emergency response. In this report, we assessed immunogenicity and, for the first time, toxicity, biodistribution, and protective efficacy in preclinical models of a two-dose self-amplifying messenger RNA (SAM) vaccine, encoding a prefusion-stabilized spike antigen of SARS-CoV-2 Wuhan-Hu-1 strain and delivered by lipid nanoparticles (LNPs). In mice, one immunization with the SAM vaccine elicited a robust spike-specific antibody response, which was further boosted by a second immunization, and effectively neutralized the matched SARS-CoV-2 Wuhan strain as well as B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta) variants. High frequencies of spike-specific germinal center B, Th0/Th1 CD4, and CD8 T cell responses were observed in mice. Local tolerance, potential systemic toxicity, and biodistribution of the vaccine were characterized in rats. In hamsters, the vaccine candidate was well-tolerated, markedly reduced viral load in the upper and lower airways, and protected animals against disease in a dose-dependent manner, with no evidence of disease enhancement following SARS-CoV-2 challenge. Therefore, the SARS-CoV-2 SAM (LNP) vaccine candidate has a favorable safety profile, elicits robust protective immune responses against multiple SARS-CoV-2 variants, and has been advanced to phase 1 clinical evaluation (NCT04758962)., Competing Interests: Declaration of interests G.M., C.P.M., J.W., T.C., G.L., K.F., L.Q., J.T.S., J.M., A.K., K.A., K-F.W., I.M., R.T., A.R., M.A.R., A-M.S., R.Jo., S.N., R.J., K.L., S.B., J.B.U., A.H.S., and D.Y. are current or former employees of the GSK group of companies and may own GSK shares and/or restricted GSK shares. G.M., J.W., L.Q., K.L., J.B.U., and D.Y. are inventors on a patent application claiming subject matter related to the SARS-CoV-2 SAM vaccine candidates described herein. P.Y.S. is a member of the Scientific Advisory Boards of AbImmune and is Founder of FlaviTech. X.X. and P.-Y.S. have filed a patent on the reverse genetic system of SARS-CoV-2. M.A.A., M.G., and C.S. received compensation from GSK to perform the rat toxicity and biodistribution assays. The other authors declare no other competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections, and vaccines are needed to treat young children and older adults. One of GSK's candidate vaccines for RSV contains recombinant RSVPreF3 protein maintained in the prefusion conformation. The differences in immune function of young children and older adults potentially require different vaccine approaches. For young children, anti-RSV immunity can be afforded during the first months of life by vaccinating the pregnant mother during the third trimester with unadjuvanted RSVPreF3, which results in protection of the infant due to the transplacental passage of anti-RSV maternal antibodies. For older adults with a waning immune response, the approach is to adjuvant the RSVPreF3 vaccine with AS01 to elicit a more robust immune response.The local and systemic effects of biweekly intramuscular injections of the RSVPreF3 vaccine (unadjuvanted, adjuvanted with AS01, or coadministered with a diphtheria-tetanus-acellular pertussis vaccine) was tested in a repeated dose toxicity study in rabbits. After three intramuscular doses, the only changes observed were those commonly related to a vaccine-elicited inflammatory reaction. Subsequently, the effects of unadjuvanted RSVPreF3 vaccine on female fertility, embryo-fetal, and postnatal development of offspring were evaluated in rats and rabbits. There were no effects on pregnancy, delivery, lactation, or the pre- and postnatal development of offspring.In conclusion, the RSVPreF3 vaccine was well-tolerated locally and systemically and was not associated with any adverse effects on female reproductive function or on the pre- and postnatal growth and development of offspring.

The novel self-amplifying mRNA (SAM) technology for vaccines consists of an engineered replication-deficient alphavirus genome encoding an RNA-dependent RNA polymerase and the gene of the target antigen. To validate the concept, the rabies glycoprotein G was chosen as antigen. The delivery system for this vaccine was a cationic nanoemulsion. To characterize the local tolerance, potential systemic toxicity and biodistribution of this vaccine, two nonclinical studies were performed. In the repeated dose toxicity study, the SAM vaccine was administered intramuscularly to rats on four occasions at two-week intervals followed by a four-week recovery period. SAM-related changes consisted of a transient increase in neutrophil count, alpha-2-macroglobulin and fibrinogen levels. Transient aspartate aminotransferase and alanine aminotransferase increases were also noted in females only. At necropsy, observations related to the elicited inflammatory reaction, such as enlargement of the draining lymph nodes were observed that were almost fully reversible by the end of the recovery period. In the biodistribution study, rats received a single intramuscular injection of SAM vaccine and then were followed until Day 60. Rabies RNA was found at the injection sites and in the draining lymph nodes one day after administration, then generally decreased in these tissues but remained detectable up to Day 60. Rabies RNA was also transiently found in blood, lungs, spleen and liver. No microscopic changes in the brain and spinal cord were recorded. In conclusion, these results showed that the rabies SAM vaccine was well-tolerated by the animals and supported the clinical development program., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Alan Stokes, Ornella Binazon, Michael Ringenberg and Luis-Alexander Rodriguez are employees of the GSK group of companies. AS, L-AR and MAR report ownership of GSK shares and/or restricted shares. Johanne Pion, Julius Haruna, Maude Bigras, Benoit Laffont, Guillaume Dubois, Karine Blouin and Nawel Ben Abdeljelil, are employees of Citoxlab, a Contract Research Organization contracted by GSK in the context of this study. Jamie Young is an employee of StageBio, a company subcontracted by Citoxlab for this study., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Coccidioides spp. are saprophytic, dimorphic fungi that are endemic to arid climates, are capable of infecting many species, and result in diverse clinical presentations. An indoor-housed laboratory rhesus macaque presented with weight loss and decreased activity and appetite. During the diagnostic evaluation, a bronchiolar-alveolar pattern in the cranial lung lobes, consistent with bronchopneumonia, was noted on radiographs. Given the poor prognosis, the macaque was euthanized. Confirming the radiographic assessment, gross necropsy findings included multifocal to coalescing areas of consolidation in the right and left cranial lung lobes. Microscopically, the consolidated regions were consistent with a pyogranulomatous bronchopneumonia and contained round, nonbudding, fungal yeast structures considered to be morphologically consistent with Coccidioides immitis. Culture and colony morphology results were confirmed through additional diagnostic testing. Sequencing of the D1-D2 domain of the 28S large ribosomal subunit positively matched with a known sequence specific to C. immitis. Serology for Coccidioides spp. by both latex agglutination (IgM) and immunodiffusion (IgG) was positive. In this rhesus macaque, the concordant results from histology, culture, DNA sequencing, and serology were collectively used to confirm the diagnosis of coccidioidomycosis. This animal likely acquired a latent pulmonary infection with Coccidioides months prior to arrival, when housed outdoors in a Coccidioides-endemic area. The nonspecific clinical presentation in this macaque, coupled with the recent history of indoor housing and lag between clinical presentation and outdoor housing, can make similar diagnostic cases challenging and highlights the need for awareness regarding animal source when making an accurate diagnosis in an institutional laboratory setting.

CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4+ and CD8+ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo.

Unlabelled: Purpose- This study assessed the pharmacological effect of a novel selective C-C chemokine receptor (CCR) 2 antagonist (GSK1344386B) on monocyte/macrophage infiltration into atherosclerotic plaque using magnetic resonance imaging (MRI) in an atherosclerotic mouse model., Methods and Results: Apolipoprotein E(-/-) mice expressing human CCR2 were fed a Western diet (vehicle group) or a Western diet plus10 mg/kg per day of GSK1344386B (GSK1344386B group). After the baseline MRI, mice were implanted with osmotic pumps containing angiotensin II, 1000 ng/kg per minute, to accelerate lesion formation. After five weeks of angiotensin II administration, mice received ultrasmall superparamagnetic iron oxide, an MRI contrast agent for the assessment of monocyte/macrophage infiltration to the plaque, and underwent imaging. After imaging, mice were euthanized, and the heart and aorta were harvested for ex vivo MRI and histopathological examination. After 5 weeks of dietary dosing, there were no significant differences between groups in body or liver weight or plasma cholesterol concentrations. An in vivo MRI reflected a decrease in ultrasmall superparamagnetic iron oxide contrast agent uptake in the aortic arch of the GSK1344386B group (P<0.05). An ex vivo MRI of the aortic root also reflected decreased ultrasmall superparamagnetic iron oxide uptake in the GSK1344386B group and was verified by absolute iron analysis (P<0.05). Although there was no difference in aortic root lesion area between groups, there was a 30% reduction in macrophage area observed in the GSK1344386B group (P<0.05)., Conclusions: An MRI was used to noninvasively assess the decreased macrophage content in the atherosclerotic plaque after selective CCR2 inhibition.

A variety of pathogens or commensals use at least one of four distinct mechanisms for decorating their surfaces with sialic acid as a strategy to avoid, subvert or inhibit host innate immunity. The metabolism of sialic acid thus is central to a range of host-pathogen interactions. The first committed step in this process, the production of free N-acetylmannosamine (ManNAc), has not been defined. Here we show that ManNAc-6-phosphate (ManNAc-6-P) is not an obligate sialate precursor in Escherichia coli K1. This conclusion was supported by 31P NMR spectroscopy of E. coli K1 derivatives engineered with different combinations of mutations in nanA (sialate aldolase or lyase), nanK (ManNAc kinase), nanE (ManNAc-6-P 2-epimerase), neuS (polysialyltransferase) and neuB (sialate synthase). The product specificities for purified NanK and NanE were determined by chromatographic analyses. Direct biochemical analysis showed that ManNAc-6-P was stable in a nanE mutant extract. The combined results indicate that neither ManNAc-6-P nor specific or non-specific phosphatase are necessary to generate the requisite ManNAc for sialate biosynthesis. Our results imply that the neuC gene product encodes an UDP-N-acetylglucosamine 2-epimerase that generates ManNAc directly from the dinucleotide-sugar precursor despite detection of only this enzyme's UDP-GlcNAc hydrolase activity. This study describes the first use of NMR for analysing intermediate flux within the sialate biosynthetic pathway.

A meningeal osteosarcoma was diagnosed in a dog displaying neurologic signs compatible with a space-occupying cerebellar lesion. Gross lesions, restricted to the brain, consisted of a solitary, compressive mass attached to the dura mater overlying the left cerebellum. The mass was composed of single and multinucleated, atypical polygonal cells that lined or rested within lacuna surrounded by eosinophilic, mineralized matrix. The matrical component stained dark green-yellow to blue with Movat's pentachrome stain, deep blue to red with Heidenhain aniline blue stain, and brown-black with Von Kossa stain. Results of these stains were interpreted as tumor osteoid. Foci of dural mineralization and osseous metaplasia were present at the point of tumor attachment. The microscopic observations were interpreted as an osteosarcoma of extraskeletal origin. To our knowledge, these findings represent the first documented case of a meningeal osteosarcoma in a domestic animal species.