Mucosal vaccine-induced cross-reactive CD8+ T cells protect against SARS-CoV-2 XBB.1.5 respiratory tract infection.

A nasally delivered chimpanzee adenoviral-vectored severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine (ChAd-SARS-CoV-2-S) is currently used in India (iNCOVACC). Here, we update this vaccine by creating ChAd-SARS-CoV-2-BA.5-S, which encodes a prefusion-stabilized BA.5 spike protein. Whereas serum neutralizing antibody responses induced by monovalent or bivalent adenoviral vaccines were poor against the antigenically distant XBB.1.5 strain and insufficient to protect in passive transfer experiments, mucosal antibody and cross-reactive memory T cell responses were robust, and protection was evident against WA1/2020 D614G and Omicron variants BQ.1.1 and XBB.1.5 in mice and hamsters. However, depletion of memory CD8+ T cells before XBB.1.5 challenge resulted in loss of protection against upper and lower respiratory tract infection. Thus, nasally delivered vaccines stimulate mucosal immunity against emerging SARS-CoV-2 strains, and cross-reactive memory CD8+ T cells mediate protection against lung infection by antigenically distant strains in the setting of low serum levels of cross-reactive neutralizing antibodies.

On-Surface Synthesis of a Radical 2D Supramolecular Organic Framework.

The design of supramolecular organic radical cages and frameworks is one of the main challenges in supramolecular chemistry. Their interesting material properties and wide applications make them very promising for (photo)redox catalysis, sensors, or host-guest spin-spin interactions. However, the high reactivity of radical organic systems makes the design of such supramolecular radical assemblies challenging. Here, we report the on-surface synthesis of a purely organic supramolecular radical framework on Au(111), by combining supramolecular and on-surface chemistry. We employ a tripodal precursor, functionalized with 7-azaindole groups that, catalyzed by a single gold atom on the surface, forms a radical molecular product constituted by a π-extended fluoradene-based radical core. The radical products self-assemble through hydrogen bonding, leading to extended 2D domains ordered in a Kagome-honeycomb lattice. This approach demonstrates the potential of on-surface synthesis for developing 2D supramolecular radical organic chemistry.

Engineering a Novel Modular Adenoviral mRNA Delivery Platform Based on Tag/Catcher Bioconjugation.

mRNA vaccines have attracted widespread research attention with clear advantages in terms of molecular flexibility, rapid development, and potential for personalization. However, current mRNA vaccine platforms have not been optimized for induction of CD4/CD8 T cell responses. In addition, the mucosal administration of mRNA based on lipid nanoparticle technology faces challenges in clinical translation. In contrast, adenovirus-based vaccines induce strong T cell responses and have been approved for intranasal delivery. To leverage the inherent strengths of both the mRNA and adenovirus platforms, we developed a novel modular adenoviral mRNA delivery platform based on Tag/Catcher bioconjugation. Specifically, we engineered adenoviral vectors integrating Tag/Catcher proteins at specific locales on the Ad capsid proteins, allowing us to anchor mRNA to the surface of engineered Ad viruses. In proof-of-concept studies, the Ad-mRNA platform successfully mediated mRNA delivery and could be optimized via the highly flexible modular design of both the Ad-mRNA and protein bioconjugation systems.

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.

Phase III Pivotal comparative clinical trial of intranasal (iNCOVACC) and intramuscular COVID 19 vaccine (Covaxin®).

One of the most preferable characteristics for a COVID-19 vaccine candidate is the ability to reduce transmission and infection of SARS-CoV-2, in addition to disease prevention. Unlike intramuscular vaccines, intranasal COVID-19 vaccines may offer this by generating mucosal immunity. In this open-label, randomised, multicentre, phase 3 clinical trial (CTRI/2022/02/40065; ClinicalTrials.gov: NCT05522335), healthy adults were randomised to receive two doses, 28 days apart, of either intranasal adenoviral vectored SARS-CoV-2 vaccine (BBV154) or licensed intramuscular vaccine, Covaxin®. Between April 16 and June 4, 2022, we enrolled 3160 subjects of whom, 2971 received 2 doses of BBV154 and 161 received Covaxin. On Day 42, 14 days after the second dose, BBV154 induced significant serum neutralization antibody titers against the ancestral (Wuhan) virus, which met the pre-defined superiority criterion for BBV154 over Covaxin®. Further, both vaccines showed cross protection against Omicron BA.5 variant. Salivary IgA titers were found to be higher in BBV154. In addition, extensive evaluation of T cell immunity revealed comparable responses in both cohorts due to prior infection. However, BBV154 showed significantly more ancestral specific IgA-secreting plasmablasts, post vaccination, whereas Covaxin recipients showed significant Omicron specific IgA-secreting plasmablasts only at day 42. Both vaccines were well tolerated. Overall reported solicited reactions were 6.9% and 25.5% and unsolicited reactions were 1.2% and 3.1% in BBV154 and Covaxin® participants respectively.

Adenoviral vectors infect B lymphocytes in vivo.

B cells are the antibody-producing arm of the adaptive immune system and play a critical role in controlling pathogens. Several groups have now demonstrated the feasibility of using engineered B cells as a therapy, including infectious disease control and gene therapy of serum deficiencies. These studies have largely utilized ex vivo modification of the cells. Direct in vivo engineering would be of utility to the field, particularly in infectious disease control where the infrastructure needs of ex vivo cell modification would make a broad vaccination campaign highly challenging. In this study we demonstrate that engineered adenoviral vectors are capable of efficiently transducing murine and human primary B cells both ex vivo and in vivo. We found that unmodified human adenovirus C5 was capable of infecting B cells in vivo, likely due to interactions between the virus penton base protein and integrins. We further describe vector modification with B cell-specific gene promoters and successfully restrict transgene expression to B cells, resulting in a strong reduction in gene expression from the liver, the main site of human adenovirus C5 infection in vivo.

Two-Dimensional Self-Assembly Driven by Intermolecular Hydrogen Bonding in Benzodi-7-azaindole Molecules on Au(111).

The control of molecular structures at the nanoscale plays a critical role in the development of materials and applications. The adsorption of a polyheteroaromatic molecule with hydrogen bond donor and acceptor sites integrated in the conjugated structure itself, namely, benzodi-7-azaindole (BDAI), has been studied on Au(111). Intermolecular hydrogen bonding determines the formation of highly organized linear structures where surface chirality, resulting from the 2D confinement of the centrosymmetric molecules, is observed. Moreover, the structural features of the BDAI molecule lead to the formation of two differentiated arrangements with extended brick-wall and herringbone packing. A comprehensive experimental study that combines scanning tunneling microscopy, high-resolution X-ray photoelectron spectroscopy, near-edge X-ray absorption fine structure spectroscopy, and density functional theory theoretical calculations has been performed to fully characterize the 2D hydrogen-bonded domains and the on-surface thermal stability of the physisorbed material.

In Vitro and In Vivo Efficacy of a Stroma-Targeted, Tumor Microenvironment Responsive Oncolytic Adenovirus in Different Preclinical Models of Cancer.

More than one million women are diagnosed annually worldwide with a gynecological cancer. Most gynecological cancers are diagnosed at a late stage, either because a lack of symptoms, such as in ovarian cancer or limited accessibility to primary prevention in low-resource countries, such as in cervical cancer. Here, we extend the studies of AR2011, a stroma-targeted and tumor microenvironment responsive oncolytic adenovirus (OAdV), whose replication is driven by a triple hybrid promoter. We show that AR2011 was able to replicate and lyse in vitro fresh explants obtained from human ovarian cancer, uterine cancer, and cervical cancer. AR2011 was also able to strongly inhibit the in vitro growth of ovarian malignant cells obtained from human ascites fluid. The virus could synergize in vitro with cisplatin even on ascites-derived cells obtained from patients heavily pretreated with neoadjuvant chemotherapy. AR2011(h404), a dual transcriptionally targeted derived virus armed with hCD40L and h41BBL under the regulation of the hTERT promoter, showed a strong efficacy in vivo both on subcutaneous and intraperitoneally established human ovarian cancer in nude mice. Preliminary studies in an immunocompetent murine tumor model showed that AR2011(m404) expressing the murine cytokines was able to induce an abscopal effect. The present studies suggest that AR2011(h404) is a likely candidate as a novel medicine for intraperitoneal disseminated ovarian cancer.

A bivalent ChAd nasal vaccine protects against SARS-CoV-2 BQ.1.1 and XBB.1.5 infection and disease in mice and hamsters.

We previously described a nasally delivered monovalent adenoviral-vectored SARS-CoV-2 vaccine (ChAd-SARS-CoV-2-S, targeting Wuhan-1 spike [S]; iNCOVACC®) that is currently used in India as a primary or booster immunization. Here, we updated the mucosal vaccine for Omicron variants by creating ChAd-SARS-CoV-2-BA.5-S, which encodes for a pre-fusion and surface-stabilized S protein of the BA.5 strain, and then tested monovalent and bivalent vaccines for efficacy against circulating variants including BQ.1.1 and XBB.1.5. Whereas monovalent ChAd-vectored vaccines effectively induced systemic and mucosal antibody responses against matched strains, the bivalent ChAd-vectored vaccine elicited greater breadth. However, serum neutralizing antibody responses induced by both monovalent and bivalent vaccines were poor against the antigenically distant XBB.1.5 Omicron strain and did not protect in passive transfer experiments. Nonetheless, nasally delivered bivalent ChAd-vectored vaccines induced robust antibody and spike-specific memory T cell responses in the respiratory mucosa, and conferred protection against WA1/2020 D614G and Omicron variants BQ.1.1 and XBB.1.5 in the upper and lower respiratory tracts of both mice and hamsters. Our data suggest that a nasally delivered bivalent adenoviral-vectored vaccine induces protective mucosal and systemic immunity against historical and emerging SARS-CoV-2 strains without requiring high levels of serum neutralizing antibody.

2D-Self-Assembled Organic Materials in Undoped Hole Transport Bilayers for Efficient Inverted Perovskite Solar Cells.

Interfaces between photoactive perovskite layer and selective contacts play a key role in the performance of perovskite solar cells (PSCs). The properties of the interface can be modified by the introduction of molecular interlayers between the halide perovskite and the transporting layers. Herein, two novel structurally related molecules, 1,3,5-tris(α-carbolin-6-yl)benzene (TACB) and the hexamethylated derivative of truxenotris(7-azaindole) (TTAI), are reported. Both molecules have the ability to self-assemble through reciprocal hydrogen bond interactions, but they have different degrees of conformational freedom. The benefits of combining these tripodal 2D-self-assembled small molecular materials with well-known hole transporting layers (HTLs), such as PEDOT:PSS and PTAA, in PSCs with inverted configuration are described. The use of these molecules, particularly the more rigid TTAI, enhanced the charge extraction efficiency and reduced the charge recombination. Consequently, an improved photovoltaic performance was achieved in comparison to the devices fabricated with the standard HTLs.

Synthetic Biology Design as a Paradigm Shift toward Manufacturing Affordable Adeno-Associated Virus Gene Therapies.

Gene therapy has demonstrated enormous potential for changing how we combat disease. By directly engineering the genetic composition of cells, it provides a broad range of options for improving human health. Adeno-associated viruses (AAVs) represent a leading gene therapy vector and are expected to address a wide range of conditions in the coming decade. Three AAV therapies have already been approved by the FDA to treat Leber's congenital amaurosis, spinal muscular atrophy, and hemophilia B. Yet these therapies cost around $850,000, $2,100,000, and $3,500,000, respectively. Such prices limit the broad applicability of AAV gene therapy and make it inaccessible to most patients. Much of this problem arises from the high manufacturing costs of AAVs. At the same time, the field of synthetic biology has grown rapidly and has displayed a special aptitude for addressing biomanufacturing problems. Here, we discuss emerging efforts to apply synthetic biology design to decrease the price of AAV production, and we propose that such efforts could play a major role in making gene therapy much more widely accessible.

In vivo editing of the pan-endothelium by immunity evading simian adenoviral vector.

Biological applications deriving from the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 site-specific nuclease system continue to impact and accelerate gene therapy strategies. Safe and effective in vivo co-delivery of the CRISPR/Cas9 system to target somatic cells is essential in the clinical therapeutic context. Both non-viral and viral vector systems have been applied for this delivery matter. Despite elegant proof-of-principle studies, available vector technologies still face challenges that restrict the application of CRISPR/Cas9-facilitated gene therapy. Of note, the mandated co-delivery of the gene-editing components must be accomplished in the potential presence of pre-formed anti-vector immunity. Additionally, methods must be sought to limit the potential of off-target editing. To this end, we have exploited the molecular promiscuities of adenovirus (Ad) to address the key requirements of CRISPR/Cas9-facilitated gene therapy. In this regard, we have endeavored capsid engineering of a simian (chimpanzee) adenovirus isolate 36 (SAd36) to achieve targeted modifications of vector tropism. The SAd36 vector with the myeloid cell-binding peptide (MBP) incorporated in the capsid has allowed selective in vivo modifications of the vascular endothelium. Importantly, vascular endothelium can serve as an effective non-hepatic cellular source of deficient serum factors relevant to several inherited genetic disorders. In addition to allowing for re-directed tropism, capsid engineering of nonhuman primate Ads provide the means to circumvent pre-formed vector immunity. Herein we have generated a SAd36. MBP vector that can serve as a single intravenously administered agent allowing effective and selective in vivo editing for endothelial target cells of the mouse spleen, brain and kidney. DATA AVAILABILITY: The data that support the findings of this study are available from the corresponding author upon reasonable request.

Bi-allelic CAMSAP1 variants cause a clinically recognizable neuronal migration disorder.

Non-centrosomal microtubules are essential cytoskeletal filaments that are important for neurite formation, axonal transport, and neuronal migration. They require stabilization by microtubule minus-end-targeting proteins including the CAMSAP family of molecules. Using exome sequencing on samples from five unrelated families, we show that bi-allelic CAMSAP1 loss-of-function variants cause a clinically recognizable, syndromic neuronal migration disorder. The cardinal clinical features of the syndrome include a characteristic craniofacial appearance, primary microcephaly, severe neurodevelopmental delay, cortical visual impairment, and seizures. The neuroradiological phenotype comprises a highly recognizable combination of classic lissencephaly with a posterior more severe than anterior gradient similar to PAFAH1B1(LIS1)-related lissencephaly and severe hypoplasia or absence of the corpus callosum; dysplasia of the basal ganglia, hippocampus, and midbrain; and cerebellar hypodysplasia, similar to the tubulinopathies, a group of monogenic tubulin-associated disorders of cortical dysgenesis. Neural cell rosette lineages derived from affected individuals displayed findings consistent with these phenotypes, including abnormal morphology, decreased cell proliferation, and neuronal differentiation. Camsap1-null mice displayed increased perinatal mortality, and RNAScope studies identified high expression levels in the brain throughout neurogenesis and in facial structures, consistent with the mouse and human neurodevelopmental and craniofacial phenotypes. Together our findings confirm a fundamental role of CAMSAP1 in neuronal migration and brain development and define bi-allelic variants as a cause of a clinically distinct neurodevelopmental disorder in humans and mice.

A Novel Piggyback Strategy for mRNA Delivery Exploiting Adenovirus Entry Biology.

Molecular therapies exploiting mRNA vectors embody enormous potential, as evidenced by the utility of this technology for the context of the COVID-19 pandemic. Nonetheless, broad implementation of these promising strategies has been restricted by the limited repertoires of delivery vehicles capable of mRNA transport. On this basis, we explored a strategy based on exploiting the well characterized entry biology of adenovirus. To this end, we studied an adenovirus-polylysine (AdpL) that embodied "piggyback" transport of the mRNA on the capsid exterior of adenovirus. We hypothesized that the efficient steps of Ad binding, receptor-mediated entry, and capsid-mediated endosome escape could provide an effective pathway for transport of mRNA to the cellular cytosol for transgene expression. Our studies confirmed that AdpL could mediate effective gene transfer of mRNA vectors in vitro and in vivo. Facets of this method may offer key utilities to actualize the promise of mRNA-based therapeutics.

Efficient Genome Editing Achieved via Plug-and-Play Adenovirus Piggyback Transport of Cas9/gRNA Complex on Viral Capsid Surface.

The capacity to efficiently deliver the gene-editing enzyme complex to target cells is favored over other forms of gene delivery as it offers one-time hit-and-run gene editing, thus improving precision and safety and reducing potential immunogenicity against edited cells in clinical applications. Here we performed a proof-of-mechanism study and demonstrated that a simian adenoviral vector for DNA delivery can be repurposed as a robust intracellular delivery platform for a functional Cas9/guide RNA (gRNA) complex to recipient cells. In this system, the clinically relevant adenovirus was genetically engineered with a plug-and-display technology based on SpyTag003/SpyCatcher003 coupling chemistry. Under physiological conditions, an off-the-shelf mixture of viral vector with SpyTag003 incorporated into surface capsid proteins and Cas9 fused with SpyCatcher003 led to a rapid titration reaction yielding adenovirus carrying Cas9SpyCatcher003 on the virus surface. The Cas9 fusion protein-conjugated viruses in the presence of a reporter gRNA delivered gene-editing functions to cells with an efficiency comparable to that of a commercial CRISPR/Cas9 transfection reagent. Our data fully validate the adenoviral "piggyback" approach to deliver an intracellularly acting enzyme cargo and, thus, warrant the prospect of engineering tissue-targeted adenovirus carrying Cas9/gRNA for in vivo gene editing.

Convection Enhanced Delivery of the Oncolytic Adenovirus Delta24-RGD in Patients with Recurrent GBM: A Phase I Clinical Trial Including Correlative Studies.

PURPOSE: Testing safety of Delta24-RGD (DNX-2401), an oncolytic adenovirus, locally delivered by convection enhanced delivery (CED) in tumor and surrounding brain of patients with recurrent glioblastoma. PATIENTS AND METHODS: Dose-escalation phase I study with 3+3 cohorts, dosing 107 to 1 × 1011 viral particles (vp) in 20 patients. Besides clinical parameters, adverse events, and radiologic findings, blood, cerebrospinal fluid (CSF), brain interstitial fluid, and excreta were sampled over time and analyzed for presence of immune response, viral replication, distribution, and shedding. RESULTS: Of 20 enrolled patients, 19 received the oncolytic adenovirus Delta24-RGD, which was found to be safe and feasible. Four patients demonstrated tumor response on MRI, one with complete regression and still alive after 8 years. Most serious adverse events were attributed to increased intracranial pressure caused by either an inflammatory reaction responding to steroid treatment or viral meningitis being transient and self-limiting. Often viral DNA concentrations in CSF increased over time, peaking after 2 to 4 weeks and remaining up to 3 months. Concomitantly Th1- and Th2-associated cytokine levels and numbers of CD3+ T and natural killer cells increased. Posttreatment tumor specimens revealed increased numbers of macrophages and CD4+ and CD8+ T cells. No evidence of viral shedding in excreta was observed. CONCLUSIONS: CED of Delta24-RGD not only in the tumor but also in surrounding brain is safe, induces a local inflammatory reaction, and shows promising clinical responses.

Potential benefits of structured lipids in bulk compound chocolate: Insights on bioavailability and effect on serum lipids.

The bioavailability impact of serum lipids in compound chocolate products based on structured lipids was studied. Compound chocolate products containing fat with and without structured lipids were digested in vitro under simulated gastrointestinal lipolysis conditions and were studied in vivo in healthy C57BL/6J mice. The in vitro digestion results show that products containing structured lipids, milk compound chocolate filling and white compound coating, significantly reduced the release rate of Free Fatty Acids (FFA) and improved the caloric reduction between 12.49% and 13.71% compared to products without structured lipids, suggesting that FFA were not absorbed. Animal feeding studies revealed no adverse effects on the compound products intake; in fact, these products reduced total cholesterol, LDL-c, VLDL-c and triacylglycerols. The present work shows the relevance of developing functional compound chocolate as providing a potential healthy initiative through the biological effect of the bioactive ingredients incorporated.

Multiple Treatment Cycles of Neural Stem Cell Delivered Oncolytic Adenovirus for the Treatment of Glioblastoma.

Tumor tropic neural stem cells (NSCs) can improve the anti-tumor efficacy of oncovirotherapy agents by protecting them from rapid clearance by the immune system and delivering them to multiple distant tumor sites. We recently completed a first-in-human trial assessing the safety of a single intracerebral dose of NSC-delivered CRAd-Survivin-pk7 (NSC.CRAd-S-pk7) combined with radiation and chemotherapy in newly diagnosed high-grade glioma patients. The maximum feasible dose was determined to be 150 million NSC.CRAd-Sp-k7 (1.875 × 1011 viral particles). Higher doses were not assessed due to volume limitations for intracerebral administration and the inability to further concentrate the study agent. It is possible that therapeutic efficacy could be maximized by administering even higher doses. Here, we report IND-enabling studies in which an improvement in treatment efficacy is achieved in immunocompetent mice by administering multiple treatment cycles intracerebrally. The results imply that pre-existing immunity does not preclude therapeutic benefits attainable by administering multiple rounds of an oncolytic adenovirus directly into the brain.