Supermassive black holes in galaxy centres can grow by the accretion of gas, liberating energy that might regulate star formation on galaxy-wide scales. The nature of the gaseous fuel reservoirs that ...power black hole growth is nevertheless largely unconstrained by observations, and is instead routinely simplified as a smooth, spherical inflow of very hot gas. Recent theory and simulations instead predict that accretion can be dominated by a stochastic, clumpy distribution of very cold molecular clouds--a departure from the 'hot mode' accretion model--although unambiguous observational support for this prediction remains elusive. Here we report observations that reveal a cold, clumpy accretion flow towards a supermassive black hole fuel reservoir in the nucleus of the Abell 2597 Brightest Cluster Galaxy (BCG), a nearby (redshift z = 0.0821) giant elliptical galaxy surrounded by a dense halo of hot plasma. Under the right conditions, thermal instabilities produce a rain of cold clouds that fall towards the galaxy's centre, sustaining star formation amid a kiloparsec-scale molecular nebula that is found at its core. The observations show that these cold clouds also fuel black hole accretion, revealing 'shadows' cast by the molecular clouds as they move inward at about 300 kilometres per second towards the active supermassive black hole, which serves as a bright backlight. Corroborating evidence from prior observations of warmer atomic gas at extremely high spatial resolution, along with simple arguments based on geometry and probability, indicate that these clouds are within the innermost hundred parsecs of the black hole, and falling closer towards it.
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IJS, KISLJ, NUK, SBMB, UL, UM, UPUK
Fibroblast activation protein (FAP) is overexpressed in cancer-associated fibroblasts and is an interesting target for cancer immune therapy, with prior studies indicating a potential to affect the ...tumor stroma. Our aim was to extend this earlier work through the development of a novel FAP immunogen with improved capacity to break tolerance for use in combination with tumor antigen vaccines.
We used a synthetic consensus (SynCon) sequence approach to provide MHC class II help to support breaking of tolerance. We evaluated immune responses and antitumor activity of this novel FAP vaccine in preclinical studies, and correlated these findings to patient data.
This SynCon FAP DNA vaccine was capable of breaking tolerance and inducing both CD8
and CD4
immune responses. In genetically diverse, outbred mice, the SynCon FAP DNA vaccine was superior at breaking tolerance compared with a native mouse FAP immunogen. In several tumor models, the SynCon FAP DNA vaccine synergized with other tumor antigen-specific DNA vaccines to enhance antitumor immunity. Evaluation of the tumor microenvironment showed increased CD8
T-cell infiltration and a decreased macrophage infiltration driven by FAP immunization. We extended this to patient data from The Cancer Genome Atlas, where we find high FAP expression correlates with high macrophage and low CD8
T-cell infiltration.
These results suggest that immune therapy targeting tumor antigens in combination with a microconsensus FAP vaccine provides two-fisted punch-inducing responses that target both the tumor microenvironment and tumor cells directly.
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Studies of interleukin (IL)-33 reveal a number of pleiotropic properties. Here, we report that IL-33 has immunoadjuvant effects in a human papilloma virus (HPV)-associated model for cancer ...immunotherapy where cell-mediated immunity is critical for protection. Two biologically active isoforms of IL-33 exist that are full-length or mature, but the ability of either isoform to function as a vaccine adjuvant that influences CD4 T helper 1 or CD8 T-cell immune responses is not defined. We showed that both IL-33 isoforms are capable of enhancing potent antigen-specific effector and memory T-cell immunity in vivo in a DNA vaccine setting. In addition, although both IL-33 isoforms drove robust IFN-γ responses, neither elevated secretion of IL-4 or immunoglobulin E levels. Further, both isoforms augmented vaccine-induced antigen-specific polyfunctional CD4(+) and CD8(+) T-cell responses, with a large proportion of CD8(+) T cells undergoing plurifunctional cytolytic degranulation. Therapeutic studies indicated that vaccination with either IL-33 isoform in conjunction with an HPV DNA vaccine caused rapid and complete regressions in vivo. Moreover, IL-33 could expand the magnitude of antigen-specific CD8(+) T-cell responses and elicit effector-memory CD8(+) T cells. Taken together, our results support the development of these IL-33 isoforms as immunoadjuvants in vaccinations against pathogens, including in the context of antitumor immunotherapy.
Vaccination remains one of the greatest medical breakthroughs in human history and has resulted in the near eradication of many formerly lethal diseases in many countries, including the complete ...eradication of smallpox. However, there remain a number of diseases for which there are no or only partially effective vaccines. There are numerous hurdles in vaccine development, of which knowing the appropriate immune response to target is one of them. Recently, tissue-resident T cells have been shown to mediate high levels of protection for several infections, although the best way to induce these cells is still unclear. Here we compare the ability to generate skin-resident T cells in sites distant from the immunization site following intramuscular and intradermal injection using optimized synthetic DNA vaccines. We found that mice immunized intradermally with a synthetic consensus DNA HIV envelope vaccine by electroporation (EP) are better able to maintain durable antigen-specific cellular responses in the skin than mice immunized by the intramuscular route. We extended these studies by delivering a synthetic DNA vaccine encoding
glycosomal phosphoenolpyruvate carboxykinase (PEPCK) by EP and again found that the intradermal route was superior to the intramuscular route for generating skin-resident PEPCK-specific T cells. We observed that when challenged with
parasites, mice immunized intradermally exhibited significant protection, while mice immunized intramuscularly did not. The protection seen in intradermally vaccinated mice supports the viability of this platform not only to generate skin-resident T cells but also to promote durable protective immune responses at relevant tissue sites.
The impact of broad-spectrum antibiotics on antimicrobial resistance and disruption of the beneficial microbiome compels the urgent investigation of bacteria-specific approaches such as ...antibody-based strategies. Among these, DNA-delivered monoclonal antibodies (DMAbs), produced by muscle cells in vivo, potentially allow the prevention or treatment of bacterial infections circumventing some of the hurdles of protein IgG delivery. Here, we optimize DNA-delivered monoclonal antibodies consisting of two potent human IgG clones, including a non-natural bispecific IgG1 candidate, targeting Pseudomonas aeruginosa. The DNA-delivered monoclonal antibodies exhibit indistinguishable potency compared to bioprocessed IgG and protect against lethal pneumonia in mice. The DNA-delivered monoclonal antibodies decrease bacterial colonization of organs and exhibit enhanced adjunctive activity in combination with antibiotics. These studies support DNA-delivered monoclonal antibodies delivery as a potential strategy to augment the host immune response to prevent serious bacterial infections, and represent a significant advancement toward broader practical delivery of monoclonal antibody immunotherapeutics for additional infectious pathogens.DNA-delivered monoclonal antibodies (DMAbs) can be produced by muscle cells in vivo, potentially allowing prevention or treatment of infectious diseases. Here, the authors show that two DMAbs targeting Pseudomonas aeruginosa proteins confer protection against lethal pneumonia in mice.
Antibody-based immune therapies targeting the T-cell checkpoint molecules CTLA-4 and PD-1 have affected cancer therapy. However, this immune therapy requires complex manufacturing and frequent ...dosing, limiting the global use of this treatment. Here, we focused on the development of a DNA-encoded monoclonal antibody (DMAb) approach for delivery of anti-CTLA-4 monoclonal antibodies
With this technology, engineered and formulated DMAb plasmids encoding IgG inserts were directly injected into muscle and delivered intracellularly by electroporation, leading to
expression and secretion of the encoded IgG. DMAb expression from a single dose can continue for several months without the need for repeated administration. Delivery of an optimized DMAb encoding anti-mouse CTLA-4 IgG resulted in high serum levels of the antibody as well as tumor regression in Sa1N and CT26 tumor models. DNA-delivery of the anti-human CTLA-4 antibodies ipilimumab and tremelimumab in mice achieved potent peak levels of approximately 85 and 58 μg/mL, respectively. These DMAb exhibited prolonged expression, with maintenance of serum levels at or above 15 μg/mL for over a year. Anti-human CTLA-4 DMAbs produced
bound to human CTLA-4 protein expressed on stimulated human peripheral blood mononuclear cells and induced T-cell activation in a functional assay
In summary, direct
expression of DMAb encoding checkpoint inhibitors serves as a novel tool for immunotherapy that could significantly improve availability and provide broader access to such therapies.
DNA-encoded monoclonal antibodies represent a novel technology for delivery and expression of immune checkpoint blockade antibodies, thus expanding patient access to, and possible clinical applications of, these therapies.
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Immune checkpoint blockade antibodies are setting a new standard of care for cancer patients. It is therefore important to assess any new immune-based therapies in the context of immune checkpoint ...blockade. Here, we evaluate the impact of combining a synthetic consensus TERT DNA vaccine that has improved capacity to break tolerance with immune checkpoint inhibitors. We observed that blockade of CTLA-4 or, to a lesser extent, PD-1 synergized with TERT vaccine, generating more robust anti-tumor activity compared to checkpoint alone or vaccine alone. Despite this anti-tumor synergy, none of these immune checkpoint therapies showed improvement in TERT antigen-specific immune responses in tumor-bearing mice. αCTLA-4 therapy enhanced the frequency of T-bet+/CD44+ effector CD8+ T cells within the tumor and decreased the frequency of regulatory T cells within the tumor, but not in peripheral blood. CTLA-4 blockade synergized more than Treg depletion with TERT DNA vaccine, suggesting that the effect of CTLA-4 blockade is more likely due to the expansion of effector T cells in the tumor rather than a reduction in the frequency of Tregs. These results suggest that immune checkpoint inhibitors function to alter the immune regulatory environment to synergize with DNA vaccines, rather than boosting antigen-specific responses at the site of vaccination.
In this issue of Molecular Therapy, Duperret et al. show synergy of DNA vaccines with immune checkpoint blockade antibodies targeting CTLA-4 and PD-1 in reducing tumor burden in mice. Importantly, these antibodies did not boost systemic immune responses and instead altered immune activation in the tumor.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
HIV Envelope (Env) is the main vaccine target for induction of neutralizing antibodies. Stabilizing Env into native-like trimer (NLT) conformations is required for recombinant protein immunogens to ...induce autologous neutralizing antibodies(nAbs) against difficult to neutralize HIV strains (tier-2) in rabbits and non-human primates. Immunizations of mice with NLTs have generally failed to induce tier-2 nAbs. Here, we show that DNA-encoded NLTs fold properly in vivo and induce autologous tier-2 nAbs in mice. DNA-encoded NLTs also uniquely induce both CD4 + and CD8 + T-cell responses as compared to corresponding protein immunizations. Murine neutralizing antibodies are identified with an advanced sequencing technology. The structure of an Env-Ab (C05) complex, as determined by cryo-EM, identifies a previously undescribed neutralizing Env C3/V5 epitope. Beyond potential functional immunity gains, DNA vaccines permit in vivo folding of structured antigens and provide significant cost and speed advantages for enabling rapid evaluation of new HIV vaccines.
Interventions to prevent HIV-1 infection and alternative tools in HIV cure therapy remain pressing goals. Recently, numerous broadly neutralizing HIV-1 monoclonal antibodies (bNAbs) have been ...developed that possess the characteristics necessary for potential prophylactic or therapeutic approaches. However, formulation complexities, especially for multiantibody deliveries, long infusion times, and production issues could limit the use of these bNAbs when deployed, globally affecting their potential application. Here, we describe an approach utilizing synthetic DNA-encoded monoclonal antibodies (dmAbs) for direct in vivo production of prespecified neutralizing activity. We designed 16 different bNAbs as dmAb cassettes and studied their activity in small and large animals. Sera from animals administered dmAbs neutralized multiple HIV-1 isolates with activity similar to that of their parental recombinant mAbs. Delivery of multiple dmAbs to a single animal led to increased neutralization breadth. Two dmAbs, PGDM1400 and PGT121, were advanced into nonhuman primates for study. High peak-circulating levels (between 6 and 34 μg/ml) of these dmAbs were measured, and the sera of all animals displayed broad neutralizing activity. The dmAb approach provides an important local delivery platform for the in vivo generation of HIV-1 bNAbs and for other infectious disease antibodies.
Synthetically engineered DNA-encoded monoclonal antibodies (DMAbs) are an in vivo platform for evaluation and delivery of human mAb to control against infectious disease. Here, we engineer DMAbs ...encoding potent anti-Zaire ebolavirus (EBOV) glycoprotein (GP) mAbs isolated from Ebola virus disease survivors. We demonstrate the development of a human IgG1 DMAb platform for in vivo EBOV-GP mAb delivery and evaluation in a mouse model. Using this approach, we show that DMAb-11 and DMAb-34 exhibit functional and molecular profiles comparable to recombinant mAb, have a wide window of expression, and provide rapid protection against lethal mouse-adapted EBOV challenge. The DMAb platform represents a simple, rapid, and reproducible approach for evaluating the activity of mAb during clinical development. DMAbs have the potential to be a mAb delivery system, which may be advantageous for protection against highly pathogenic infectious diseases, like EBOV, in resource-limited and other challenging settings.
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•DMAbs are an in vivo approach for mAb development/delivery against Zaire ebolavirus•DMAbs can be functionally equivalent to recombinant mAb•Reproducible and cost-effective mouse model for in vivo mAb evaluation•Enables evaluation of fully human mAbs rapidly in vivo
Monoclonal antibodies are an important approach for emerging infectious disease prevention. Patel et al. demonstrate engineering and in vivo delivery of DNA-encoded monoclonal antibodies (DMAbs) targeting the Zaire ebolavirus (EBOV) glycoprotein. DMAbs protect against lethal mouse-adapted EBOV and are useful for rapid evaluation of fully human mAbs in live animal models.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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