Anti-PD1 blockade benefits only a subset of patients with head and neck squamous cell carcinoma (HNSCC), highlighting the need for approaches that overcome tumor immune resistance. Here, using the doggybone DNA (dbDNA™) platform, we developed CaVac OPT, an optimized dual-antigen DNA vaccine targeting MAGED4B and FJX1, which are overexpressed in most HPV-negative HNSCC and multiple solid tumors. In the MOC-2 oral cancer model, CaVac OPT significantly reduced tumor growth and when combined with anti-PD1 therapy, further delayed progression and improved survival. Immune profiling showed increased infiltration of CD4+ and CD8+ T cells, expansion of stem-like Tcf1+ populations, without increase in regulatory T cells, a reduced M2/M1 macrophage ratio and activation of interferon gamma associated pathways with suppression of tumor-promoting signals. These findings demonstrate that CaVac OPT reprograms the tumor microenvironment, converting cold HNSCC into T cell-inflamed responsive tumors. CaVac OPT represents a promising strategy for achieving durable control of aggressive, immunotherapy-resistant head and neck cancer. ### Competing Interest Statement N.S., C.H.O., G.J.T., S.C.C., C.W. and K.P. are the authors of the patent covering cancer vaccine targeting MAGED4B and FJX1 (WO2021074655A1) Touchlight University of Liverpool, https://ror.org/04xs57h96

Influenza virus represents a challenge for traditional vaccine approaches due to its seasonal changes and potential for zoonotic transmission. Nucleic acid vaccines can overcome some of these challenges, especially through the inclusion of multiple antigens to increase the breadth of response. RNA vaccines were an important part of the response to the COVID-19 pandemic, but for future outbreaks DNA vaccines may have some advantages in terms of stability and manufacturing cost that warrant continuing investigation to fully realize their potential. Here, we investigate influenza virus vaccines made using a closed linear DNA platform, Doggybone™ DNA (dbDNA), produced by a rapid and scalable cell-free method. Influenza vaccines have mostly focussed on Haemagglutinin (HA), but the inclusion of Neuraminidase (NA) may provide additional protection. Here, we explored the potential of including NA in a dbDNA vaccine, looking at DNA optimization, mechanism and breadth of protection. We showed that DNA targeting sequences (DTS) improved immune responses against HA but not NA. We explored whether NA vaccine-induced protection against influenza virus infection was cell-mediated, but depletion of CD8 and NK cells made no impact, suggesting it was antibody-mediated. This is reflected in the restriction of protection to homologous strains of influenza virus. Importantly, we saw that including both HA and NA in a single combined vaccine did not dampen the immune response to either one. Overall, we show that linear dbDNA can induce an immune response against NA, which may offer increased protection in instances of HA mismatch where NA remains more conserved.

Alternatives are needed to remove the pain, injury, cross‐infection, and hazardous waste associated with needle and syringe (N+S)‐based vaccination. Reported here is the use of novel ultrasound‐responsive protein cavitation nuclei (pCaN), formed using the model antigen bovine serum albumin (BSA), to achieve effective transcutaneous delivery. Upon exposure to ultrasound (US), these pCaN instigate cavitation events which propel themselves and co‐located DNA vectors into the skin. US parameters as well as pCaN and DNA concentration are refined to achieve optimal expression of encoded luciferase transgene. Twenty‐four hours post‐treatment, luciferase expression in the skin, by IVIS imaging, was 1.67 × 10 6 ±941943, photons per sec for N+S intradermal injection and 1.49 × 10 6 ±261832 for cavitation‐mediated delivery ( p >0.05). Hence, there is no significant difference in luciferase level achieved, but improved homogeneity and reproducibility of expression are evident in mice treated using US‐mediated cavitation. Despite this equivalence in luciferase levels, a >5× higher level ( p <0.02) of anti‐luciferase antibodies is achieved when cavitation is used versus N+S injection. Antibody levels against BSA, resulting from the use of BSA pCaN, are equivalent for the two groups. PCaN can be formed from a range of antigenic proteins and DNA can encode a range of antigenic proteins, so this approach has wide‐ranging implications for needle‐free vaccination.

To combat the COVID-19 pandemic, an assortment of vaccines has been developed. Nucleic acid vaccines have the advantage of rapid production, as they only require a viral antigen sequence and can readily be modified to detected viral mutations. Doggybone™ DNA vaccines targeting the spike protein of SARS-CoV-2 have been generated and compared with a traditionally manufactured, bacterially derived plasmid DNA vaccine that utilizes the same spike sequence. Administered to Syrian hamsters by jet injection at two dose levels, the immunogenicity of both DNA vaccines was compared following two vaccinations. Immunized hamsters were then immunosuppressed and exposed to SARS-CoV-2. Significant differences in body weight were observed during acute infection, and lungs collected at the time of euthanasia had significantly reduced viral RNA, infectious virus, and pathology compared with irrelevant DNA-vaccinated controls. Moreover, immune serum from vaccinated animals was capable of neutralizing SARS-CoV-2 variants of interest and importance in vitro. These data demonstrate the efficacy of a synthetic DNA vaccine approach to protect hamsters from SARS-CoV-2.

Background Tissue resident memory T (Trm) cells act as sentinels and early responders to infection. (RSV)-specific Trm cells have been detected in the lungs after human RSV infection, but their role is unknown. To dissect the protective function of Trm cells, mice were infected with RSV; infected mice developed antigen-specific CD8+ Trm cells in the lungs and airways. Intranasally transferring cells from the airways of previously infected animals to naïve animals reduced weight loss on infection in the recipient mice. Transfer of airway CD8 cells led to reduced disease and viral load in recipient mice. Because DNA vaccines induce a systemic T-cell response, we compared vaccination with infection. Intramuscular DNA immunization induced RSV-specific CD8 T cells, but they were not protective. Infection but not immunization induced antigen-specific Trm cells in a range of tissues. These findings demonstrate a protective role for airway CD8 against RSV and support the need for vaccines to induce antigen-specific airway cells. Objectives In this study the role of airway resident memory T cells (Trm) in protection against RSV infection was examined. It was hypothesised that since Trms are present at the site of infection and can act as sentinels, their induction would lead to rapid pathogen clearance. Methods Antigen specific Trm cells (CD69+CD103+) were characterized in the lungs and other tissues ofmice after infection or vaccination. For vaccination, DNA was delivered intramuscularly in a prime-boost-boost regimen separated by two weeks. To dissect the protective function of Trm, cells were obtained by bronchoalveolar lavage from RSV experienced mice, sorted and transferred intranasally into naïve animals prior to intranasal challenge a day later with RSV. Results 1. Prior RSV infection protects against subsequent infection and induces both RSV specific antibody and CD8 T cells. Naïve or previously RSV infected mice were infected with RSV intranasally, weight was measured daily. Viral load was measured on day 4 after infection. Anti-RSV antibody responses were measured on day 7 after infection. 2. Airway cells, not splenocytes from RSV experienced mice can protect from disease in the absence of antibody. Mice received cells from airways and spleens of RSV exposed or PBS exposed mice intranasally prior to infection with RSV. Viral load was measured on day 7. 3. Immunisation and infection induce different antigen specific T cell populations in different tissues. Mice were either vaccinated with RSV DNA vaccine or intranasally infected with RSV. After 8 weeks all mice were sacrificed. The left and right flank skin and muscle, blood, spleen, airway and lung cells were analysed. RSV specific CD8+ T cells and proportion of Trm were assessed. Conclusions RSV infection leads to CD8+ Trm formation in the lungs and the airways of mice. Intra-airway cell transfer of RSV experienced CD4 or CD8 cells to naïve animals protects from disease upon challenge in the absence of antibodies. RSV infection leads to dissemination of RSV specific CD8+ Trm cells however this is not reproduced by IM vaccination. Localised delivery of vaccines to the lungs may be needed to generate formation of Trm in the airways.

The application of induced pluripotent stem cells (iPSCs) in advanced therapies is increasing at pace, but concerns remain over their clinical safety profile. We report the first-ever application of doggybone DNA (dbDNA) vectors to generate human iPSCs. dbDNA vectors are closed-capped linear double-stranded DNA gene expression cassettes that contain no bacterial DNA and are amplified by a chemically defined, current good manufacturing practice (cGMP)-compliant methodology. We achieved comparable iPSC reprogramming efficiencies using transiently expressing dbDNA vectors with the same iPSC reprogramming coding sequences as the state-of-the-art OriP/EBNA1 episomal vectors but, crucially, in the absence of p53 shRNA repression. Moreover, persistent expression of EBNA1 from bacterially derived episomes resulted in stimulation of the interferon response, elevated DNA damage, and increased spontaneous differentiation. These cellular activities were diminished or absent in dbDNA-iPSCs, resulting in lines with a greater stability and safety potential for cell therapy.

CD19-specific chimeric antigen receptor (CAR19) T cells, generated using viral vectors, are an efficacious but costly treatment for B cell malignancies. The nonviral piggyBac transposon system provides a simple and inexpensive alternative for CAR19 T cell production. Until now, piggyBac has been plasmid based, facilitating economical vector amplification in bacteria. However, amplified plasmids have several undesirable qualities for clinical translation, including bacterial genetic elements, antibiotic-resistance genes, and the requirement for purification to remove endotoxin. Doggybones (dbDNA) are linear, covalently closed, minimal DNA vectors that can be inexpensively produced enzymatically in vitro at large scale. Importantly, they lack the undesirable features of plasmids. We used dbDNA incorporating piggyBac to generate CAR19 T cells. Initially, expression of functional transposase was evident, but stable CAR expression did not occur. After excluding other causes, additional random DNA flanking the transposon within the dbDNA was introduced, promoting stable CAR expression comparable to that of using plasmid components. Our findings demonstrate that dbDNA incorporating piggyBac can be used to generate CAR T cells and indicate that there is a requirement for DNA flanking the piggyBac transposon to enable effective transposition. dbDNA may further reduce the cost and improve the safety of CAR T cell production with transposon systems.

The manufacture of large quantities of high-quality DNA is a major bottleneck in the production of viral vectors for gene therapy. Touchlight Genetics has developed a proprietary abiological technology that addresses the major issues in commercial DNA supply. The technology uses 'rolling-circle' amplification to produce large quantities of concatameric DNA that is then processed to create closed linear double-stranded DNA by enzymatic digestion. This novel form of DNA, Doggybone™ DNA (dbDNA™), is structurally distinct from plasmid DNA. Here we compare lentiviral vectors production from dbDNA™ and plasmid DNA. Lentiviral vectors were administered to neonatal mice via intracerebroventricular injection. Luciferase expression was quantified in conscious mice continually by whole-body bioluminescent imaging. We observed long-term luciferase expression using dbDNA™-derived vectors, which was comparable to plasmid-derived lentivirus vectors. Here we have demonstrated that functional lentiviral vectors can be produced using the novel dbDNA™ configuration for delivery in vitro and in vivo. Importantly, this could enable lentiviral vector packaging of complex DNA sequences that have previously been incompatible with bacterial propagation systems, as dbDNA™ technology could circumvent such restrictions through its phi29-based rolling-circle amplification.

Vaccination with DNA that encodes cancer antigens is a simple and convenient way to raise immunity against cancer and has already shown promise in the clinical setting. Conventional plasmid DNA is commonly used which together with the encoded antigen also includes bacterial immunostimulatory CpG motifs to target the DNA sensor Toll-like receptor 9. Recently DNA vaccines using doggybone DNA (dbDNA™), have been developed without the use of bacteria. The cell-free process relies on the use of Phi29 DNA polymerase to amplify the template followed by protelomerase TelN to complete individual closed linear DNA. The resulting DNA contains the required antigenic sequence, a promoter and a poly A tail but lacks bacterial sequences such as an antibiotic resistance gene, prompting the question of immunogenicity. Here we compared the ability of doggybone DNA vaccine with plasmid DNA vaccine to induce adaptive immunity using clinically relevant oncotargets E6 and E7 from HPV. We demonstrate that despite the inability to trigger TLR9, doggybone DNA was able to induce similar levels of cellular and humoral immunity as plasmid DNA, with suppression of established TC-1 tumours.

Tissue resident memory T (Trm) cells act as sentinels and early responders to infection. Respiratory syncytial virus (RSV)-specific Trm cells have been detected in the lungs after human RSV infection, but whether they have a protective role is unknown. To dissect the protective function of Trm cells, BALB/c mice were infected with RSV; infected mice developed antigen-specific CD8+ Trm cells (CD103+/CD69+) in the lungs and airways. Intranasally transferring cells from the airways of previously infected animals to naïve animals reduced weight loss on infection in the recipient mice. Transfer of airway CD8 cells led to reduced disease and viral load and increased interferon-γ in the airways of recipient mice, while CD4 transfer reduced tumor necrosis factor-α in the airways. Because DNA vaccines induce a systemic T-cell response, we compared vaccination with infection for the effect of memory CD8 cells generated in different compartments. Intramuscular DNA immunization induced RSV-specific CD8 T cells, but they were immunopathogenic and not protective. Notably, there was a marked difference in the induction of Trm cells; infection but not immunization induced antigen-specific Trm cells in a range of tissues. These findings demonstrate a protective role for airway CD8 against RSV and support the need for vaccines to induce antigen-specific airway cells.

Recombinant adeno-associated virus (AAV) represents one of the most promising delivery vehicles for genetic medicines. However, the manufacture of plasmid DNA for the production of AAV presents a number of significant challenges, including scalability, fidelity, mis-incorporation of plasmid-derived DNA sequences, high costs and long lead times for GMP production. Touchlight has developed a novel, rapid, in vitro, enzymatic technology for multi-gram scale GMP manufacture of DNA that addresses all of the issues of DNA manufacture for AAV production. The process combines the use of two enzymes; Phi29 DNA polymerase and a protelomerase to generate covalently closed, linear DNA constructs known as doggyboneTM DNA or dbDNATM. The process is rapid, cost effective, of high fidelity and eliminates antibiotic resistance genes. Here we present a case study, with data generated by Cobra Biologics as part of an on-going Innovate UK-funded collaboration, demonstrating that AAV particles can be produced using dbDNATM with total and genomic titres equivalent to AAV particles made with plasmid DNA. In parallel experiments, with an academic collaborator, we have seen in vivo expression of reporter genes after administration of AAV vectors manufactured using dbDNATM (unpublished data). As such, we believe that dbDNATM has the potential to resolve a number of significant challenges in the production of AAV vectors at both clinical and commercial scale.

DNA vaccines can be manufactured cheaply, easily and rapidly and have performed well in pre-clinical animal studies. However, clinical trials have so far been disappointing, failing to evoke a strong immune response, possibly due to poor antigen expression. To improve antigen expression, improved technology to monitor DNA vaccine transfection efficiency is required. In the current study, we compared plasmid encoded tdTomato, mCherry, Katushka, tdKatushka2 and luciferase as reporter proteins for whole animal in vivo imaging. The intramuscular, subcutaneous and tattooing routes were compared and electroporation was used to enhance expression. We observed that overall, fluorescent proteins were not a good tool to assess expression from DNA plasmids, with a highly heterogeneous response between animals. Of the proteins used, intramuscular delivery of DNA encoding either tdTomato or luciferase gave the clearest signal, with some Katushka and tdKatushka2 signal observed. Subcutaneous delivery was weakly visible and nothing was observed following DNA tattooing. DNA encoding haemagglutinin was used to determine whether immune responses mirrored visible expression levels. A protective immune response against H1N1 influenza was induced by all routes, even after a single dose of DNA, though qualitative differences were observed, with tattooing leading to high antibody responses and subcutaneous DNA leading to high CD8 responses. We conclude that of the reporter proteins used, expression from DNA plasmids can best be assessed using tdTomato or luciferase. But, the disconnect between visible expression level and immunogenicity suggests that in vivo whole animal imaging of fluorescent proteins has limited utility for predicting DNA vaccine efficacy.

Nucleic acid-based vaccines (NAVs) are a promising alternative to conventional influenza vaccines with the potential to increase influenza vaccine availability due to their simplicity in design and rapid speed of production. NAVs can also target multiple influenza antigens and control flu variants. Traditionally NAVs have been DNA plasmids however, we are continuing to explore new methods that may enhance vaccine efficacy. Recently new focus has been on RNA cassettes as NAVs. RNA vaccines combine conceptual advantages in that they focus on delivery of only the coding cassette. However, RNA vaccines have a short half-life and cause interferon-induced fevers. Here we describe a new NAV approach where we study delivery of a linear DNA cassette [Doggybone linear closed DNA [(dbDNA)] produced by an enzymatic process that yields an antigen expression cassette comprising a promoter, DNA antigen, poly A tail, and telomeric ends. This focused approach has many of the advantages of plasmid DNA as well as a minimal cassette size similar to RNA strategies. For this study, we characterized the specific CD4(+) and CD8(+) T cell responses and determined the hemagglutination inhibition (HI) titers induced by dbDNA and compared the responses with those of an optimized plasmid DNA (pDNA) vaccine encoding the same H1N1 influenza A/PR/8/34HA gene. Immunizations with the constructs resulted in similar humoral and cellular immune responses. Both constructs induced high-titer HI antibodies and fully protected animals from lethal viral challenge. The data obtained from this study provides important validation for further development of novel vector approaches.

The use of DNA to deliver vaccine antigens offers many advantages, including ease of manufacture and cost. However, most DNA vaccines are plasmids and must be grown in bacterial culture, necessitating elements that are either unnecessary for effective gene delivery (for example, bacterial origins of replication) or undesirable (for example, antibiotic resistance genes). Removing these elements may improve the safety profile of DNA for the delivery of vaccines. Here, we describe a novel, double-stranded, linear DNA construct produced by an enzymatic process that solely encodes an antigen expression cassette, comprising antigen, promoter, polyA tail and telomeric ends. We compared these constructs (called 'Doggybones' because of their shape) with conventional plasmid DNA. Using luciferase-expressing constructs, we demonstrated that expression levels were equivalent between Doggybones and plasmids both in vitro and in vivo. When mice were immunized with DNA constructs expressing the HIV envelope protein gp140, equivalent humoral and cellular responses were induced. Immunizations with either construct type expressing hemagluttinin were protective against H1N1 influenza challenge. This is the first example of an effective DNA vaccine, which can be produced on a large scale by enzymatic processes.