Safe and efficient nucleic acid delivery to targeted cell populations remains a challenge in the fields of cell and gene therapy. Toward this end, we attempted to utilize the "DogTag-DogCatcher" system to target adenoviral vectors. "DogTag" is a short peptide that forms a spontaneous isopeptide bond upon mixing with its partner protein, "DogCatcher." We genetically incorporated the DogTag peptide into the protein responsible for initial binding of the virus to its target cell, the fiber. This allowed permanent linking of DogCatcher-fused single-domain or single-chain antibodies at the fiber. This modification allowed simple, effective, and exclusive targeting of the vector to cells bound by the linked antibody. This enhanced gene transfer into primary B and T cells by up to 60-fold in vitro and 2- to 3-fold in vivo in mice without other alterations to vector tropism. Although the system's in vivo performance is currently suboptimal and additional engineering is needed prior to further use, these studies form the basis of a novel method for targeting adenovirus that can be combined with additional well-characterized adenovirus modifications toward applications in cell engineering, gene therapy, vaccines, oncolytics, and others.

Safe and efficient nucleic acid delivery to targeted cell populations remains a significant unmet need in the fields of cell and gene therapy. Towards this end, we pursued Adenoviral vectors genetically modified with the "DogTag" molecular glue peptide, which forms a spontaneous covalent bond with its partner protein, "DogCatcher". Genetic fusion of DogCatcher to single-domain or single-chain antibodies allowed covalent tethering of the antibody at defined locales on the vector capsid. This modification allowed simple, effective and exclusive targeting of the vector to cells bound by the linked antibody. This dramatically enhanced gene transfer into primary B and T cells in vitro and in vivo in mice. These studies form the basis of a novel method for targeting Adenovirus that is functional in stringent in vivo contexts and can be combined with additional well characterized Adenovirus modifications towards applications in cell engineering, gene therapy, vaccines, oncolytics, and others.

There is an unmet need for new therapies to promote mucosal healing and restoration of epithelial barrier function in inflammatory bowel disease (IBD). Major therapeutics for IBD act systemically; however, the development of novel approaches to specifically deliver a therapy to the gut has the theoretical advantage of optimizing benefit while limiting side effects. Adenoviral (Ad) gene therapy has been successful in treating cancer and has recently been used in clinical trials as a vaccine for SARS-Cov2. Ad vectors can be modified to target specific tissues and may be more effective vehicles for therapeutics than other systemic drugs. Gene therapy has promise to achieve these goals in IBD, and we developed an approach to deliver IL-22 via an Ad vector. IL-22 is important in maintaining the epithelial barrier at mucosal sites like the skin, airway, and intestine. Epithelial cells are the primary target of IL-22 signaling, where it activates transcription of genes involved in cell proliferation and promotes antimicrobial functions. We hypothesize that gene therapy with Ad vectors to deliver Il22 directly to the intestine could be an effective therapeutic for IBD by promoting intestinal wound healing and microbial homeostasis. To test this hypothesis, we injected human adenovirus serotype 5 (huAd5) expressing Il22 (Ad-CMV-Il22) intravenously into 6-week-old C57BL/6 mice. IL-22 protein levels in the serum, liver, ileum, and colon were increased with Ad-CMV-Il22 compared to control (Ad-CMV-GFP). We found increased transcription of genes downstream of IL-22 signaling, including Lcn2, Mt1, and Mt2, in the liver, colon, and ileum. Ad-CMV-Il22 induced increased crypt depth and goblet cell count in the ileum and colon, and increased villus length in the ileum compared to Ad-CMV-GFP. Changes to the stool microbiome included increased abundance of the pro-tolerogenic microbiota with Ad-CMV-Il22 in both colon and ileum. It is known that huAd5 has liver tropism and gene therapy with hAdv5 has been associated with liver toxicity. In line with this, we observed higher levels of IL-22 in the liver than in the serum, ileum, or colon after Ad-CMV-Il22 treatment. To reduce liver tropism, we modified the vector to incorporate myeloid-binding peptide (MBP) on the virus fiber, which re-targets the virus to the intestine and vascular endothelium. Incorporation of MBP succeeded in reducing IL-22 protein levels in the liver. Increased colon and ileal crypt depth and villus length were seen with Ad-CMV-MBP-Il22 compared with untargeted virus. Together, these data suggest that IL-22 gene therapy in the intestine can promote cell proliferation and microbial homeostasis. In the future, this adenoviral gene therapy could be modified further to increase Il22 gene delivery specifically to intestinal tissues.

Chikungunya virus (CHIKV) is a pathogenic arthritogenic alphavirus responsible for large-scale human epidemics for which a vaccine was recently approved for use. Mayaro virus (MAYV) is a related emerging alphavirus with epidemic potential with circulation overlap potential with CHIKV. We previously reported the ability of a non-replicating human adenovirus (AdV)-vectored vaccine expressing the MAYV structural polyprotein to protect against disease in mice following challenge with MAYV, CHIKV and UNAV. Herein, we evaluated mouse immunity and protective efficacy for an AdV-CHIKV full structural polyprotein vaccine in combination with heterologous AdV-MAYV prime/boost regimens versus vaccine coadministration. Heterologous prime/boost regimens skewed immunity toward the prime vaccine antigen but allowed for a boost of cross-neutralizing antibodies, while vaccine co-administration elicited robust, balanced responses capable of boosting. All immunization strategies protected against disease from homologous virus infection, but reciprocal protective immunity differences were revealed upon challenge with heterologous viruses. In vivo passive transfer experiments reproduced the inequity in reciprocal cross-protection after heterologous MAYV challenge. We detected in vitro antibody-dependent enhancement of MAYV replication, suggesting a potential mechanism for the lack of cross-protection. Our findings provide important insights into rational alphavirus vaccine design that may have important implications for the evolving alphavirus vaccine landscape.

A mucosal route of vaccination could prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication at the site of infection and limit transmission. We compared protection against heterologous XBB.1.16 challenge in nonhuman primates (NHPs) ~5 months following intramuscular boosting with bivalent mRNA encoding WA1 and BA.5 spike proteins or mucosal boosting with a WA1-BA.5 bivalent chimpanzee adenoviral-vectored vaccine delivered by intranasal or aerosol device. NHPs boosted by either mucosal route had minimal virus replication in the nose and lungs, respectively. By contrast, protection by intramuscular mRNA was limited to the lower airways. The mucosally delivered vaccine elicited durable airway IgG and IgA responses and, unlike the intramuscular mRNA vaccine, induced spike-specific B cells in the lungs. IgG, IgA and T cell responses correlated with protection in the lungs, whereas mucosal IgA alone correlated with upper airway protection. This study highlights differential mucosal and serum correlates of protection and how mucosal vaccines can durably prevent infection against SARS-CoV-2.

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.

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.

PDF file - 81K, Supplementary Table 2 Primers and probe sets utilized in this study were designed by the Primer Express 1.5 software and synthesized by Sigma-Aldrich. These primers and probes were utilized for quantitative PCR in ancillary biologic studies, and the data

Supplementary Figure 1, Table 1 from Mechanism by Which Mcl-1 Regulates Cancer-Specific Apoptosis Triggered by mda-7/IL-24, an IL-10–Related Cytokine

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.Graphical abstract:

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.

PDF file - 51K, Patients were then assigned into one of three Ad5.SSTR/TK.RGD dose cohorts ranging from 1 x 109 vp/d to 1 x 1012 vp/d (6 x 107 pfu/d to 6 x 1010 pfu/d). This dose administration strategy has been validated in our preclinical studies; assigned Ad5.SSTR/TK

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.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy).This article has been retracted at the request of the editor-in-chief. Similarities were found between images within this article. Image analysis performed by the editorial office confirmed findings of image duplication in Figures 1B, 4, and 8A. This reuse (and in part misrepresentation) of data without appropriate attribution represents a severe abuse of the scientific publishing system. No authors responded when contacted about the retraction.

Objective Exploring how public health nurses (PHNs) provide community-based support to women who are refugees and mothering. Design A constructivist grounded theory (CGT) design was used where intersectionality as an analytical tool was applied. Varying data collection approaches including focus groups were used. Sample Twelve PHNs from four public health units in Western Canada participated in this study. Results Participants in this study described an overall process of creating safe relational space to address a basic social problem of establishing trust while managing structural forces. This overarching process was expressed through burning with passion, connecting while looking beyond, protecting from re-traumatization, and fostering independence. Findings reveal strategies PHNs used to enhance health equity. This study extends critical caring theory to include sociopolitical and economic influences on public health nursing practice. Consequences of these influences on the mothering refugee women population are also revealed. Implications include structural integration of trauma-and-violence-informed principles to support public health nursing practice. Conclusions This study adds to an emerging body of knowledge on PHNs work with complex populations. Innovative application of intersectionality is demonstrated as an effective approach to analyzing impacts of broad sociopolitical priorities on communities that are systemically marginalized.

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.