Adenoviral vectors have demonstrated great potential for gene therapy of many diseases. Significant effort has been devoted in this field to achieve targeted gene delivery to specific cells with the intention of improving the therapeutic index and safety. These targeting strategies require a powerful vector detection system to assess vector function. Present methods used to localize vectors are inadequate for evaluating targeting activity. Post-transduction reporter gene expression cannot accurately represent the physical biodistribution of vectors; vector labeling with chemical substrates may affect the infectivity of virions; and probing for viral components requires extensive preparation and strong accessible signal. The field of virotherapy has further challenged the effectiveness of current vector detection systems. Due to the dynamic nature of replicative agents, current static detection techniques cannot be used to monitor replication and spread of these viruses, two key functions of the ideal replicative agent. With respect to the former issue of vector detection in targeting schemes, we sought to develop a vector labeling approach to directly detect adenoviral particles. We designed a genetic labeling system that would also fulfill the latter goal of dynamic monitoring of adenoviral replication and spread. We hypothesized that an adenoviral structural protein genetically fused to an imaging ligand would allow vector detection and serve as a signature of viral replication and localization. Recently we successfully generated and characterized a recombinant adenovirus labeled with capsid fusion pIX-EGFP. The fluorescent label minimally perturbed viral function and provided an optical property useful for vector detection in tracking of cellular infection, flow cytometry, and tissue sections following systemic administration. We expanded our labeling strategy further to produce bioluminescent adenoviruses. The rationale for this pursuit revolves around the fact that bioluminescence can be detected much deeper in tissues than fluorescent proteins with much greater sensitivity and dynamic range as well as virtually no background luminescence. We generated bioluminescent adenoviruses by incorporating Renilla luciferase (RL) and firefly luciferase (FL) onto the pIX locale. Although these two proteins were significantly larger than EGFP, both Ad-IX-RL and Ad-IX-FL were rescued. After propagation and CsCl centrifugation of these two viruses, the viral gradients were fractionated and analyzed for viral DNA content and luciferase activity. Bioluminescence peaks corresponding with the viral DNA were detected for the mature (bottom) and empty capsid (top) viral bands of Ad-IX-RL and Ad-IX-FL, indicating the retained activity of both luciferases not only in the context of a fusion with pIX but also physical association with the virus capsid. We are characterizing these vectors further and are evaluating their general utility as well as potential for noninvasive detection in mice. Bioluminescent adenoviruses generated with our genetic labeling system will have great implications in vector targeting studies and can potentially be used to monitor adenovirus replication and spread in virotherapy applications.
ABSTRACT The utility of the present generation of adenovirus (Ad) vectors for gene therapy applications could be improved by restricting native viral tropism to selected cell types. In order to achieve modification of Ad tropism, we proposed to exploit a minor component of viral capsid, protein IX (pIX), for genetic incorporation of targeting ligands. Based on the proposed structure of pIX, we hypothesized that its C terminus could be used as a site for incorporation of heterologous peptide sequences. We engineered recombinant Ad vectors containing modified pIX carrying a carboxy-terminal Flag epitope along with a heparan sulfate binding motif consisting of either eight consecutive lysines or a polylysine sequence. Using an anti-Flag antibody, we have shown that modified pIXs are incorporated into virions and display Flag-containing C-terminal sequences on the capsid surface. In addition, both lysine octapeptide and polylysine ligands were accessible for binding to heparin-coated beads. In contrast to virus bearing lysine octapeptide, Ad vector displaying a polylysine was capable of recognizing cellular heparan sulfate receptors. We have demonstrated that incorporation of a polylysine motif into the pIX ectodomain results in a significant augmentation of Ad fiber knob-independent infection of CAR-deficient cell types. Our data suggest that the pIX ectodomain can serve as an alternative to the fiber knob, penton base, and hexon proteins for incorporation of targeting ligands for the purpose of Ad tropism modification.
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.
SUMMARY The Coronavirus Disease 2019 pandemic has made deployment of an effective vaccine a global health priority. We evaluated the protective activity of a chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike protein (ChAd-SARS-CoV-2-S) in challenge studies with Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mice expressing the human angiotensin-converting enzyme 2 receptor. Intramuscular dosing of ChAd-SARS-CoV-2-S induces robust systemic humoral and cell-mediated immune responses and protects against lung infection, inflammation, and pathology but does not confer sterilizing immunity, as evidenced by detection of viral RNA and induction of anti-nucleoprotein antibodies after SARS-CoV-2 challenge. In contrast, a single intranasal dose of ChAd-SARS-CoV-2-S induces high levels of systemic and mucosal IgA and T cell responses, completely prevents SARS-CoV-2 infection in the upper and lower respiratory tracts, and likely confers sterilizing immunity in most animals. Intranasal administration of ChAd-SARS-CoV-2-S is a candidate for preventing SARS-CoV-2 infection and transmission, and curtailing pandemic spread.
To mount a strong anti-tumor immune response, non T cell inflamed (cold) tumors may require combination treatment encompassing vaccine strategies preceding checkpoint inhibition. In vivo targeted delivery of tumor-associated antigens (TAA) to dendritic cells (DCs), relying on the natural functions of primary DCs in situ, represents an attractive vaccination strategy. In this study we made use of a full-length MART-1 expressing C/B-chimeric adenoviral vector, consisting of the Ad5 capsid and the Ad3 knob (Ad5/3), which we previously showed to selectively transduce DCs in human skin and lymph nodes. Our data demonstrate that chimeric Ad5/3 vectors encoding TAA, and able to target human DCs in situ, can be used to efficiently induce expansion of functional tumor-specific CD8⁺ effector T cells, either from a naïve T cell pool or from previously primed T cells residing in the melanoma-draining sentinel lymph nodes (SLN). These data support the use of Ad3-knob containing viruses as vaccine vehicles for in vivo delivery. "Off-the-shelf" DC-targeted Ad vaccines encoding TAA could clearly benefit future immunotherapeutic approaches.
Adenoviral vectors offer great promise for gene therapy of various diseases. Recent development in targeting gene delivery calls for a more powerful vector detection system to assess vector functionality. Current vector imaging techniques-including reporter gene expression, vector labeling, and viral component detection-are limited in several ways. The physical biodistribution of vectors cannot be accurately represented by post-infection reporter gene expression; vector labeling with chemical substrates may affect the infectivity of virions; and detection of viral components requires extensive preparation and strong accessible signal. When applied in the field of virotherapy, the drawbacks of current vector detection systems are even more evident. Due to the dynamic nature of replicative agents, current static detection techniques based on biopsy examination cannot effectively confirm replication and spread of these viruses, two key functions of the ideal replicative agent. To address the former issue of vector detection in targeting situations, we sought to develop a vector labeling approach to directly visualize adenoviral particles. We conceived of a genetically based labeling system that would also fulfill the latter purpose of dynamic monitoring of adenoviral replication and spread. We hypothesized that an adenoviral structural protein genetically fused to an imaging ligand would enable vector detection and serve as a signature of viral replication and localization. Recently we successfully generated and characterized a recombinant adenovirus labeled with capsid fusion pIX-EGFP. The fluorescent label had minimal effect on viral function and conferred an optical property useful for vector detection in tracking of cellular infection, flow cytometry, and tissue sections following systemic administration. We expanded our labeling strategy further to make red fluorescent adenoviruses. One reason for designing red fluorescent adenoviruses is that a better signal-to-noise ratio can be achieved in the red spectral range, yielding improved vector detection in tissue which typically contains high green autofluorescence. In addition, red fluorescence would provide better tissue penetration allowing deeper noninvasive detection of these red fluorescent proteins. We generated red fluorescent adenoviruses by incorporating monomeric (mRFP1) and tandem dimer (tdimer2(12)) red fluorescent proteins onto the pIX locale. After propagation and CsCl centrifugation of both viruses, red mature (bottom) and empty capsid (top) viral bands were observed. Fractionation studies validated our visual observation by revealing that both the bottom and top bands of the two viruses contained red fluorescence which co-migrated with viral DNA. Abundant purified Ad-IX-mRFP1 and Ad-IX-tdimer2(12) viral particles could be detected by fluorescence microscopy. We are characterizing these vectors further and are evaluating their general utility as well as potential for noninvasive detection in mice. Red fluorescent adenoviruses generated with our genetic labeling system will have great impact in vector targeting studies and can potentially be used to monitor adenovirus replication and spread in virotherapy applications.
Peritoneal compartmentalization of advanced stage ovarian cancer provides a rational scenario for gene therapy strategies. Several groups are exploring intraperitoneal administration of adenoviral (Ad) vectors for this purpose. We examined in vitro gene transfer in the presence of ascites fluid from ovarian cancer patients and observed significant inhibition of Ad-mediated gene transfer. The inhibitory activity was not identified as either complement or cellular factors, but depletion of IgG from ascites removed the inhibitory activity, implicating neutralizing anti-Ad antibodies. A wide range of preexisting anti-Ad antibody titers in patient ascites fluid was measured by ELISA. Western blot analysis demonstrated that the antibodies were directed primarily against the Ad fiber protein. To circumvent inhibition by neutralizing antibodies, a genetically modified adenoviral vector was tested. The Ad5Luc.RGD vector has an Arg-Gly-Asp (RGD) peptide sequence inserted into the fiber knob domain and enters cells through a nonnative pathway. Compared with the conventional Ad5 vector, Ad5Luc.RGD directed efficient gene transfer to cell lines and primary ovarian cancer cells in the presence of ascites fluid containing high-titer neutralizing anti-Ad antibodies. These results suggest that such modified Ad vectors will be needed to achieve efficient gene transfer in the clinical setting.
Abstract The application of cancer gene therapy has heretofore been restricted to local, or locoregional, neoplastic disease contexts. This is owing to the lack of gene transfer vectors, which embody the requisite target cell selectivity in vivo required for metastatic disease applications. To this end, we have explored novel vector engineering paradigms to adapt adenovirus for this purpose. Our novel strategy exploits three distinct targeting modalities that operate in functional synergy. Transcriptional targeting is achieved via the hROBO4 promoter, which restricts transgene expression to proliferative vascular endothelium. Viral binding is modified by incorporation of an RGD4C peptide in the HI loop of the fiber knob for recognition of cellular integrins. Liver sequestration is mitigated by ablation of factor X binding to the major capsid protein hexon by a serotype swap approach. The combination of these technologies into the context of a single-vector agent represents a highly original approach. Studies in a murine model of disseminated cancer validated the in vivo target cell selectivity of our vector agent. Of note, clear gains in therapeutic index accrued these vector modifications. Whereas there is universal recognition of the value of vector targeting, very few reports have validated its direct utility in the context of cancer gene therapy. In this regard, our article validates the direct gains that may accrue these methods in the stringent delivery context of disseminated neoplastic disease. Efforts to improve vector targeting thus represent a critical direction to fully realize the promise of cancer gene therapy.
Abstract TRAIL continues to garner substantial interest as a recombinant cancer therapeutic while the native cytokine itself serves important tumor surveillance functions when expressed in membrane-anchored form on activated immune effector cells. We have recently developed the genetically stabilized TRAIL platform TR3 in efforts to improve the limitations associated with currently available drug variants. While in the process of characterizing mesothelin-targeted TR3 variants using a single chain antibody (scFv) delivery format (SS-TR3), we discovered that the membrane-tethered cytokine had a substantially increased activity profile compared to non-targeted TR3. However, cell death proceeded exclusively via a bystander mechanism and protected the mesothelin-positive targets from apoptosis rather than leading to their elimination. Incorporation of a spacer-into the mesothelin surface antigen or the cancer drug itself-converted SS-TR3 into a cis-acting phenotype. Further experiments with membrane-anchored TR3 variants and the native cytokine confirmed our hypothesis that membrane-proximal TRAIL species lack the capacity to physically engage their cognate receptors coexpressed on the same cell membrane. Our findings not only provide an explanation for the “peaceful” coexistence of ligand and receptor of a representative member of the TNF superfamily but give us vital clues for the design of activity-enhanced TR3-based cancer therapeutics.
