A Small Molecule RIG-I Agonist Serves as an Adjuvant to Induce Broad Multifaceted Influenza Virus Vaccine Immunity.

Retinoic acid-inducible gene I (RIG-I) is essential for activating host cell innate immunity to regulate the immune response against many RNA viruses. We previously identified that a small molecule compound, KIN1148, led to the activation of IFN regulatory factor 3 (IRF3) and served to enhance protection against influenza A virus (IAV) A/California/04/2009 infection. We have now determined direct binding of KIN1148 to RIG-I to drive expression of IFN regulatory factor 3 and NF-κB target genes, including specific immunomodulatory cytokines and chemokines. Intriguingly, KIN1148 does not lead to ATPase activity or compete with ATP for binding but activates RIG-I to induce antiviral gene expression programs distinct from type I IFN treatment. When administered in combination with a vaccine against IAV, KIN1148 induces both neutralizing Ab and IAV-specific T cell responses compared with vaccination alone, which induces comparatively poor responses. This robust KIN1148-adjuvanted immune response protects mice from lethal A/California/04/2009 and H5N1 IAV challenge. Importantly, KIN1148 also augments human CD8+ T cell activation. Thus, we have identified a small molecule RIG-I agonist that serves as an effective adjuvant in inducing noncanonical RIG-I activation for induction of innate immune programs that enhance adaptive immune protection of antiviral vaccination.

Protective Efficacy of Gastrointestinal SARS-CoV-2 Delivery against Intranasal and Intratracheal SARS-CoV-2 Challenge in Rhesus Macaques.

Live oral vaccines have been explored for their protective efficacy against respiratory viruses, particularly for adenovirus serotypes 4 and 7. The potential of a live oral vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), however, remains unclear. In this study, we assessed the immunogenicity of live SARS-CoV-2 delivered to the gastrointestinal tract in rhesus macaques and its protective efficacy against intranasal and intratracheal SARS-CoV-2 challenge. Postpyloric administration of SARS-CoV-2 by esophagogastroduodenoscopy resulted in limited virus replication in the gastrointestinal tract and minimal to no induction of mucosal antibody titers in rectal swabs, nasal swabs, and bronchoalveolar lavage fluid. Low levels of serum neutralizing antibodies were induced and correlated with modestly diminished viral loads in nasal swabs and bronchoalveolar lavage fluid following intranasal and intratracheal SARS-CoV-2 challenge. Overall, our data show that postpyloric inoculation of live SARS-CoV-2 is weakly immunogenic and confers partial protection against respiratory SARS-CoV-2 challenge in rhesus macaques. IMPORTANCE SARS-CoV-2 remains a global threat, despite the rapid deployment but limited coverage of multiple vaccines. Alternative vaccine strategies that have favorable manufacturing timelines, greater ease of distribution, and improved coverage may offer significant public health benefits, especially in resource-limited settings. Live oral vaccines have the potential to address some of these limitations; however, no studies have yet been conducted to assess the immunogenicity and protective efficacy of a live oral vaccine against SARS-CoV-2. Here, we report that oral administration of live SARS-CoV-2 in nonhuman primates may offer prophylactic benefits, but the formulation and route of administration will require further optimization.

Characterization of a Novel Compound That Stimulates STING-Mediated Innate Immune Activity in an Allele-Specific Manner.

The innate immune response to cytosolic DNA involves transcriptional activation of type I interferons (IFN-I) and proinflammatory cytokines. This represents the culmination of intracellular signaling pathways that are initiated by pattern recognition receptors that engage DNA and require the adaptor protein Stimulator of Interferon Genes (STING). These responses lead to the generation of cellular and tissue states that impair microbial replication and facilitate the establishment of long-lived, antigen-specific adaptive immunity. Ultimately this can lead to immune-mediated protection from infection but also to the cytotoxic T cell-mediated clearance of tumor cells. Intriguingly, pharmacologic activation of STING-dependent phenotypes is known to enhance both vaccine-associated immunogenicity and immune-based anti-tumor therapies. Unfortunately, the STING protein exists as multiple variant forms in the human population that exhibit differences in their reactivity to chemical stimuli and in the intensity of molecular signaling they induce. In light of this, STING-targeting drug discovery efforts require an accounting of protein variant-specific activity. Herein we describe a small molecule termed M04 that behaves as a novel agonist of human STING. Importantly, we find that the molecule exhibits a differential ability to activate STING based on the allelic variant examined. Furthermore, while M04 is inactive in mice, expression of human STING in mouse cells rescues reactivity to the compound. Using primary human cells in ex vivo assays we were also able to show that M04 is capable of simulating innate responses important for adaptive immune activation such as cytokine secretion, dendritic cell maturation, and T cell cross-priming. Collectively, this work demonstrates the conceivable utility of a novel agonist of human STING both as a research tool for exploring STING biology and as an immune potentiating molecule.

Progesterone and a phospholipase inhibitor increase the endosomal bis(monoacylglycero)phosphate content and block HIV viral particle intercellular transmission.

Progesterone, the cationic amphiphile U18666A and a phospholipase inhibitor (Methyl Arachidonyl Fluoro Phosphonate, MAFP) inhibited by 70%-90% HIV production in viral reservoir cells, i.e. human THP-1 monocytes and monocyte-derived macrophages (MDM). These compounds triggered an inhibition of fluid phase endocytosis (macropinocytosis) and modified cellular lipid homeostasis since endosomes accumulated filipin-stained sterols and Bis(Monoacylglycero)Phosphate (BMP). BMP was quantified using a new cytometry procedure and was increased by 1.25 times with MAFP, 1.7 times with U18666A and 2.5 times with progesterone. MAFP but not progesterone or U18666A inhibited the hydrolysis of BMP by the Pancreatic Lipase Related Protein 2 (PLRP2) as shown by in-vitro experiments. The possible role of sterol transporters in steroid-mediated BMP increase is discussed. Electron microscopy showed the accumulation of viral particles either into large intracellular viral-containing compartments or outside the cells, indicating that endosomal accumulation of BMP could block intracellular biogenesis of viral particles while inhibition of macropinocytosis would prevent viral particle uptake. This is the first report linking BMP metabolism with a natural steroid such as progesterone or with involvement of a phospholipase A1 activity. BMP cellular content could be used as a biomarker for efficient anti-viral drugs.

Exosomes derived from HIV-1-infected cells contain trans-activation response element RNA.

Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 10(4)-10(6) copies/ml TAR RNA in exosomes derived from infected culture supernatants and 10(3) copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS.

Exosomes as intercellular signalosomes and pharmacological effectors.

Cell secretion is a general process involved in various biological responses. Exosomes are part of this process and have gained considerable scientific interest in the past five years. Several steps through investigations across the last 20 years can explain this interest. First characterized during reticulocyte maturation, they were next evidenced as a key player in the immune response and cancer immunotherapy. More recently they were reported as vectors of mRNAs, miRNAs and also lipid mediators able to act on target cells. They are the only type of vesicles released from an intracellular compartment from cells in viable conditions. They appear as a vectorized signaling system operating from inside a donor cell towards either the periphery, the cytosol, or possibly to the nucleus of target cells. Exosomes from normal cells trigger positive effects, whereas those from pathological ones, such as tumor cells or infected ones may trigger non-positive health effects. Therefore regulating the biogenesis and secretion of exosomes appear as a pharmacological challenge to intervene in various pathophysiologies. Exosome biogenesis and molecular content, interaction with target cells, utilisation as biomarkers, and functional effects in various pathophysiologies are considered in this review.