Transdermal microarrayed electroporation for enhanced cancer immunotherapy based on DNA vaccination.

Despite the tremendous clinical potential of nucleic acid-based vaccines, their efficacy to induce therapeutic immune response has been limited by the lack of efficient local gene delivery techniques in the human body. In this study, we develop a hydrogel-based organic electronic device (µEPO) for both transdermal delivery of nucleic acids and in vivo microarrayed cell electroporation, which is specifically oriented toward one-step transfection of DNAs in subcutaneous antigen-presenting cells (APCs) for cancer immunotherapy. The µEPO device contains an array of microneedle-shaped electrodes with pre-encapsulated dry DNAs. Upon a pressurized contact with skin tissue, the electrodes are rehydrated, electrically triggered to release DNAs, and then electroporate nearby cells, which can achieve in vivo transfection of more than 50% of the cells in the epidermal and upper dermal layer. As a proof-of-concept, the µEPO technique is employed to facilitate transdermal delivery of neoantigen genes to activate antigen-specific immune response for enhanced cancer immunotherapy based on a DNA vaccination strategy. In an ovalbumin (OVA) cancer vaccine model, we show that high-efficiency transdermal transfection of APCs with OVA-DNAs induces robust cellular and humoral immune responses, including antigen presentation and generation of IFN-γ+ cytotoxic T lymphocytes with a more than 10-fold dose sparing over existing intramuscular injection (IM) approach, and effectively inhibits tumor growth in rodent animals.

RGDK-lipopeptide for targeting genetic vaccines to antigen presenting cells.

To ensure effective immune response in genetic immunizations, DNA/mRNA vaccines need to be delivered to body's antigen presenting cells (APCs) which is a challenging task. This is primarily due to presence of high concentrations of various degradative enzymes inside them. To this end, mannose receptor (over expressed in APCs) selective cationic liposomes have been used in the past for delivering antigen-encoded plasmid DNA to APCs. APCs also express integrin receptors on their cell surfaces. However, studies aimed at delivering DNA vaccines into APCs via integrin receptors have not yet been undertaken. Herein, we report on the use of cationic liposomes of a priorly disclosed α5ß1 integrin receptor selective RGDK-lipopeptide for macrophage transfection. In this study, we have used pCMV-GFP (as model DNA vaccine) and RAW 264.7 cells (mouse macrophages cells) as model APC. We show that the liposomes of RGDK-lipopeptide containing a previously reported endosome-disrupting histidinylated lipid and DOPE (as co-lipid) in 0.5:0.5:1.0 mole ratio are the most competent in transfecting macrophage cells (44%). Findings in the fluorescence resonance energy transfer based membrane fusogencity assay revealed that the enhanced macrophage transfection efficiency of the liposomes containing RGDK-lipopeptide, endosome-disrupting histidinylated and DOPE may originate from its higher membrane fusogenicity than that for liposomes containing only RGDK-lipopeptide and DOPE. The presently described biologically safe liposomal formulations of RGDK-lipopeptide are expected to find biomedical applications in future for combating cancer and infectious diseases through genetic immunizations.

What We Learned about the Feasibility of Gene Electrotransfer for Vaccination on a Model of COVID-19 Vaccine.

DNA vaccination is one of the emerging approaches for a wide range of applications, including prophylactic vaccination against infectious diseases and therapeutic vaccination against cancer. The aim of this study was to evaluate the feasibility of our previously optimized protocols for gene electrotransfer (GET)-mediated delivery of plasmid DNA into skin and muscle tissues on a model of COVID-19 vaccine. Plasmids encoding the SARS-CoV-2 proteins spike (S) and nucleocapsid (N) were used as the antigen source, and a plasmid encoding interleukin 12 (IL-12) was used as an adjuvant. Vaccination was performed in the skin or muscle tissue of C57BL/6J mice on days 0 and 14 (boost). Two weeks after the boost, blood, spleen, and transfected tissues were collected to determine the expression of S, N, IL-12, serum interferon-γ, the induction of antigen-specific IgG antibodies, and cytotoxic T-cells. In accordance with prior in vitro experiments that indicated problems with proper expression of the S protein, vaccination with S did not induce S-specific antibodies, whereas significant induction of N-specific antibodies was detected after vaccination with N. Intramuscular vaccination outperformed skin vaccination and resulted in significant induction of humoral and cell-mediated immunity. Moreover, both boost and adjuvant were found to be redundant for the induction of an immune response. Overall, the study confirmed the feasibility of the GET for DNA vaccination and provided valuable insights into this approach.

A chimeric human/dog-DNA vaccine against CSPG4 induces immunity with therapeutic potential in comparative preclinical models of osteosarcoma.

The high mortality rate of osteosarcoma (OSA) patients highlights the requirement of alternative strategies. The young age of patients, as well as the rarity and aggressiveness of the disease, limits opportunities for the robust testing of novel therapies, suggesting the need for valuable preclinical systems. Having previously shown the overexpression of the chondroitin sulfate proteoglycan (CSPG)4 in OSA, herein the functional consequences of its downmodulation in human OSA cells were evaluated in vitro, with a significant impairment of cell proliferation, migration, and osteosphere generation. The potential of a chimeric human/dog (HuDo)-CSPG4 DNA vaccine was explored in translational comparative OSA models, including human xenograft mouse models and canine patients affected by spontaneous OSA. The adoptive transfer of HuDo-CSPG4 vaccine-induced CD8+ T cells and sera in immunodeficient human OSA-bearing mice delayed tumor growth and metastasis development. HuDo-CSPG4 vaccination resulted safe and effective in inducing anti-CSPG4 immunity in OSA-affected dogs, which displayed prolonged survival as compared to controls. Finally, HuDo-CSPG4 was also able to induce a cytotoxic response in a human surrogate setting in vitro. On the basis of these results and the high predictive value of spontaneous OSA in dogs, this study paves the way for a possible translation of this approach to humans.

Prevention of Inflammation-Driven Colon Carcinogenesis in Human MUC1 Transgenic Mice by Vaccination with MUC1 DNA and Dendritic Cells.

The preventive efficacy of MUC1-specific DNA immunization on inflammation-driven colon carcinogenesis in human MUC1 transgenic (MUC1.Tg) mice was investigated. Mice were vaccinated with MUC1 DNA mixed with autologous bone-marrow-derived dendritic cells (BMDCs), and then colonic tumors were induced by azoxymethane (AOM) injection and oral administration of dextran sulfate sodium (DSS). Two types of tumors, squamous metaplasia and tubular adenoma, were observed. Both expressed high levels of MUC1 as indicated by the binding of anti-MUC1 antibodies with different specificities, whereas MUC1 expression was not detected in normal colonic mucosa. When mice were immunized with MUC1 DNA + BMDCs, tumor incidence, tumor number, and tumor size were significantly reduced. In contrast, vaccination with MUC1 DNA alone or BMDCs alone was ineffective in reducing tumor burden. Inflammation caused by DSS was not suppressed by the MUC1 DNA + BMDCs vaccination. Furthermore, MUC1 protein expression levels, as judged by anti-MUC1 antibody binding in tumors grown after vaccination, did not significantly differ from the control. In conclusion, an inflammation-driven carcinogenesis model was established in MUC1.Tg mice, closely resembling human colon carcinogenesis. In this model, vaccination with MUC1 DNA + BMDCs was effective in overriding MUC1 tolerance and reducing the tumor burden by a mechanism not affecting the level of colonic inflammation.

Title: Immunotherapy; a ground-breaking remedy for spinal cord injury with stumbling blocks: An overview.

Spinal cord injury (SCI) is a debilitating disorder with no known standard and effective treatment. Despite its ability to exacerbate SCI sequel by accelerating auto-reactive immune cells, an immune response is also considered essential to the healing process. Therefore, immunotherapeutic strategies targeting spinal cord injuries may benefit from the dual nature of immune responses. An increasing body of research suggests that immunization against myelin inhibitors can promote axon remyelination after SCI. However, despite advancements in our understanding of neuroimmune responses, immunoregulation-based therapeutic strategies have yet to receive widespread acceptance. Therefore, it is a prerequisite to enhance the understanding of immune regulation to ensure the safety and efficacy of immunotherapeutic treatments. The objective of the present study was to provide an overview of previous studies regarding the advantages and limitations of immunotherapeutic strategies for functional recovery after spinal cord injury, especially in light of limiting factors related to DNA and cell-based vaccination strategies by providing a novel prospect to lay the foundation for future studies that will help devise a safe and effective treatment for spinal cord injury.

Plasmid DNA for Therapeutic Applications in Cancer.

Recently, the interest in using nucleic acids for therapeutic applications has been increasing. DNA molecules can be manipulated to express a gene of interest for gene therapy applications or vaccine development. Plasmid DNA can be developed to treat different diseases, such as infections and cancer. In most cancers, the immune system is limited or suppressed, allowing cancer cells to grow. DNA vaccination has demonstrated its capacity to stimulate the immune system to fight against cancer cells. Furthermore, plasmids for cancer gene therapy can direct the expression of proteins with different functions, such as enzymes, toxins, and cytotoxic or proapoptotic proteins, to directly kill cancer cells. The progress and promising results reported in animal models in recent years have led to interesting clinical results. These DNA strategies are expected to be approved for cancer treatment in the near future. This review discusses the main strategies, challenges, and future perspectives of using plasmid DNA for cancer treatment.

Gene Gun Her2/neu DNA Vaccination: Evaluation of Vaccine Efficacy in a Syngeneic Her2/neu Mouse Tumor Model.

Genetic vaccination using naked plasmid DNA is an immunization strategy both against infectious diseases and cancer.In order to improve efficacy of DNA vaccines, particularly in large animals and humans, different strategies have been pursued. These vaccination strategies are based on different application routes, schedules and coexpression of immunomodulatory molecules as adjuvants. Our mouse tumor model offers the possibility to investigate Her2/neu DNA vaccines in different settings, that is, intramuscular or intradermal application with or without coexpression of adjuvants. The immunogenicity of predicted peptides for Her2/neu specific memory T cells were screened and confirmed after intramuscular and intradermal application. Protection from tumor growth in tumor challenge experiments and both T cell and humoral immune responses against Her2/neu peptides are used as surrogate parameters for vaccine efficacy.

Pyroptosis-inducing active caspase-1 as a genetic adjuvant in anti-cancer DNA vaccination.

Pyroptosis is a recently discovered form of inflammatory programmed necrosis characterized by caspase-1-mediated and gasdermin D-dependent cell death leading to the release of pro-inflammatory cytokines such as Interleukin-1 beta (IL-1ß). Here, we evaluated whether pyroptosis could be exploited in DNA vaccination by incorporating a constitutively active variant of caspase-1 to the antigen-expressing DNA. In vitro, transfection with constitutively active caspase-1 DNA induced pro-IL-1ß maturation and IL-1ß release as well as gasdermin D-dependent cell death. To test active caspase-1 as a genetic adjuvant for the induction of antigen-specific T cell responses, mice were vaccinated intradermally with a DNA vaccine consisting of the active caspase-1 plasmid together with a plasmid encoding an ovalbumin-derived CD8 T cell epitope. Active caspase-1 accelerated and amplified antigen-specific CD8 T cell responses when administered simultaneously with the DNA vaccine at an equimolar dose. Moreover, upon challenge with melanoma cells expressing ovalbumin, mice vaccinated with the antigen vaccine adjuvanted with active caspase-1 showed significantly better survival compared to the non-adjuvanted group. In conclusion, we have developed a novel genetic adjuvant that for the first time employs the pyroptosis pathway to improve DNA vaccination against cancer.

Dominant Antiviral CD8+ T Cell Responses Empower Prophylactic Antibody-Eliciting Vaccines Against Cytomegalovirus.

Human cytomegalovirus (HCMV) is an ubiquitous herpesvirus that can cause serious morbidity and mortality in immunocompromised or immune-immature individuals. A vaccine that induces immunity to CMV in these target populations is therefore highly needed. Previous attempts to generate efficacious CMV vaccines primarily focused on the induction of humoral immunity by eliciting neutralizing antibodies. Current insights encourage that a protective immune response to HCMV might benefit from the induction of virus-specific T cells. Whether addition of antiviral T cell responses enhances the protection by antibody-eliciting vaccines is however unclear. Here, we assessed this query in mouse CMV (MCMV) infection models by developing synthetic vaccines with humoral immunity potential, and deliberately adding antiviral CD8+ T cells. To induce antibodies against MCMV, we developed a DNA vaccine encoding either full-length, membrane bound glycoprotein B (gB) or a secreted variant lacking the transmembrane and intracellular domain (secreted (s)gB). Intradermal immunization with an increasing dose schedule of sgB and booster immunization provided robust viral-specific IgG responses and viral control. Combined vaccination of the sgB DNA vaccine with synthetic long peptides (SLP)-vaccines encoding MHC class I-restricted CMV epitopes, which elicit exclusively CD8+ T cell responses, significantly enhanced antiviral immunity. Thus, the combination of antibody and CD8+ T cell-eliciting vaccines provides a collaborative improvement of humoral and cellular immunity enabling enhanced protection against CMV.

Elastin-like Polymers as Nanovaccines: Protein Engineering of Self-Assembled, Epitope-Exposing Nanoparticles.

In this chapter we describe two unconventional strategies for the formulation of new nanovaccines. Both strategies are based on obtaining chimeric genes that code for proteins in which the major antigens of the pathogens are fused to an elastin-like recombinamer (ELR) as carrier. ELRs are a family of synthetic protein biopolymers obtained using DNA recombinant techniques. The ELRs employed in the present chapter are block copolymers that are able to assemble, under controlled conditions, into nanoparticles similar to virus-like particles and to provoke an immune response. We describe the biosynthesis of ELRs genetically fused to an antigenic sequence from Mycobacterium tuberculosis and a simple procedure for obtaining stable nanoparticles displaying the antigen in the first strategy. The second approach describes the production of a DNA vaccine library consisting of plasmids codifying for major antigens from Rift Valley fever virus fused to different ELR-based block copolymer architectures.The procedures described can be adapted for the production of other chimeric DNA-protein vaccines based on protein polymer carriers.

Aquaculture Reuse Water, Genetic Line, and Vaccination Affect Rainbow Trout (Oncorhynchus mykiss) Disease Susceptibility and Infection Dynamics.

Infectious hematopoietic necrosis virus (IHNV) and Flavobacterium psychrophilum are major pathogens of farmed rainbow trout. Improved control strategies are desired but the influence of on-farm environmental factors that lead to disease outbreaks remain poorly understood. Water reuse is an important environmental factor affecting disease. Prior studies have established a replicated outdoor-tank system capable of varying the exposure to reuse water by controlling water flow from commercial trout production raceways. The goal of this research was to evaluate the effect of constant or pulsed reuse water exposure on survival, pathogen prevalence, and pathogen load. Herein, we compared two commercial lines of rainbow trout, Clear Springs Food (CSF) and Troutex (Tx) that were either vaccinated against IHNV with a DNA vaccine or sham vaccinated. Over a 27-day experimental period in constant reuse water, all fish from both lines and treatments, died while mortality in control fish in spring water was <1%. Water reuse exposure, genetic line, vaccination, and the interaction between genetic line and water exposure affected survival (P<0.05). Compared to all other water sources, fish exposed to constant reuse water had 46- to 710-fold greater risk of death (P<0.0001). Tx fish had a 2.7-fold greater risk of death compared to CSF fish in constant reuse water (P ≤ 0.001), while risk of death did not differ in spring water (P=0.98). Sham-vaccinated fish had 2.1-fold greater risk of death compared to vaccinated fish (P=0.02). Both IHNV prevalence and load were lower in vaccinated fish compared to sham-vaccinated fish, and unexpectedly, F. psychrophilum load associated with fin/gill tissues from live-sampled fish was lower in vaccinated fish compared to sham-vaccinated fish. As a result, up to forty-five percent of unvaccinated fish were naturally co-infected with F. psychrophilum and IHNV and the coinfected fish exhibited the highest IHNV loads. Under laboratory challenge conditions, co-infection with F. psychrophilum and IHNV overwhelmed IHNV vaccine-induced protection. In summary, we demonstrate that exposure to reuse water or multi-pathogen challenge can initiate complex disease dynamics that can overwhelm both vaccination and host genetic resistance.

Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs).

Plasmid DNA (pDNA) isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research. Almost all pDNA purification involves disruption of bacteria, removal of membrane lipids, proteins and genomic DNA, purification of pDNA from bulk lysate, and concentration of pDNA for downstream applications. While many liquid-phase and solid-phase pDNA purification methods are used, the final pDNA preparations are usually contaminated with varied degrees of host RNA, which cannot be completely digested by RNase A. To develop a simple, cost-effective, and yet effective method for RNA depletion, we investigated whether commercially available size selection magnetic beads (SSMBs), such as Mag-Bind® TotalPure NGS Kit (or Mag-Bind), can completely deplete bacterial RNA in pDNA preparations. In this proof-of-principle study, we demonstrated that, compared with RNase A digestion and two commercial plasmid affinity purification kits, the SSMB method was highly efficient in depleting contaminating RNA from pDNA minipreps. Gene transfection and bacterial colony formation assays revealed that pDNA purified from SSMB method had superior quality and integrity to pDNA samples cleaned up by RNase A digestion and/or commercial plasmid purification kits. We further demonstrated that the SSMB method completely depleted contaminating RNA in large-scale pDNA samples. Furthermore, the Mag-bind-based SSMB method costs only 5-10% of most commercial plasmid purification kits on a per sample basis. Thus, the reported SSMB method can be a valuable and inexpensive tool for the removal of bacterial RNA for routine pDNA preparations.

Tick-Tattoo: DNA Vaccination Against B. burgdorferi or Ixodes scapularis Tick Proteins.

Introduction: Borrelia burgdorferi sensu lato (sl) is the causative agent of Lyme borreliosis. Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines. DNA tattoo vaccination with B. afzelii strain PKo OspC in mice has proven to be fully protective against B. afzelii syringe challenge and induces a favorable humoral immunity compared to recombinant protein vaccination. Alternatively, several recombinant protein vaccines based on tick proteins have shown promising effect in tick-bite infection models. In this study, we evaluated the efficacy of DNA vaccines against Borrelia OspC or tick antigens in a tick-bite infection model. Method: We vaccinated C3H/HeN mice with OspC using a codon-optimized DNA vaccine or with recombinant protein. We challenged these mice with B. burgdorferi sensu stricto (ss)-infected Ixodes scapularis nymphs. Subsequently, we vaccinated C3H/HeN mice with DNA vaccines coding for tick proteins for which recombinant protein vaccines have previously resulted in interference with tick feeding and/or Borrelia transmission: Salp15, tHRF, TSLPI, and Tix-5. These mice were also challenged with B. burgdorferi ss infected Ixodes scapularis nymphs. Results: DNA tattoo and recombinant OspC vaccination both induced total IgG responses. Borrelia cultures and DNA loads of skin and bladder remained negative in the mice vaccinated with OspC DNA vaccination, except for one culture. DNA vaccines against tick antigens Salp15 and Tix-5 induced IgG responses, while those against tHRF and TSLPI barely induced any IgG response. In addition, Borrelia cultures, and DNA loads from mice tattooed with DNA vaccines against tick proteins TSLPI, Salp15, tHRF, and Tix-5 were all positive. Conclusion: A DNA tattoo vaccine against OspC induced high specific IgG titers and provided near total protection against B. burgdorferi ss infection by tick challenge. In contrast, DNA tattoo vaccines against tick proteins TSLPI, Salp15, tHRF, and Tix-5 induced low to moderate IgG titers and did not provide protection. Therefore, DNA tattoo vaccination does not seem a suitable vaccine strategy to identify, or screen for, tick antigens for anti-tick vaccines. However, DNA tattoo vaccination is a straightforward and effective vaccination platform to assess novel B. burgdorferi sl antigen candidates in a relevant tick challenge model.

Strategy to enhance dendritic cell-mediated DNA vaccination in the lung.

We here introduce a new paradigm to promote pulmonary DNA vaccination. Specifically, we demonstrate that nanoparticles designed to rapidly penetrate airway mucus (mucus-penetrating particle or MPP) enhance the delivery of inhaled model DNA vaccine (i.e. ovalbumin-expressing plasmids) to pulmonary dendritic cells (DC), leading to robust and durable local and trans-mucosal immunity. In contrast, mucus-impermeable particles were poorly taken up by pulmonary DC following inhalation, despite their superior ability to mediate DC uptake in vitro compared to MPP. In addition to the enhanced immunity achieved in mucosal surfaces, inhaled MPP unexpectedly provided significantly greater systemic immune responses compared to gold-standard approaches applied in the clinic for systemic vaccination, including intradermal injection and intramuscular electroporation. We also showed here that inhaled MPP significantly enhanced the survival of an orthotopic mouse model of aggressive lung cancer compared to the gold-standard approaches. Importantly, we discovered that MPP-mediated pulmonary DNA vaccination induced memory T-cell immunity, particularly the ready-to-act effector memory-biased phenotype, both locally and systemically. The findings here underscore the importance of breaching the airway mucus barrier to facilitate DNA vaccine uptake by pulmonary DC and thus to initiate full-blown immune responses.

In vivo Targeting of DNA Vaccines to Dendritic Cells via the Mannose Receptor Induces Long-Lasting Immunity against Melanoma.

Herein, we report effective, C-type lectin mannose receptor (MR)-selective, in vivo dendritic cell (DC)-targeting lipid nanoparticles (LNPs) of a novel lipid-containing mannose-mimicking di-shikimoyl- and guanidine head group and two n-hexadecyl hydrophobic tails (DSG). Subcutaneous administration of LNPs of the DSG/p-CMV-GFP complex showed a significant expression of green fluorescence protein in the CD11c+ DCs of the neighboring lymph nodes compared to the control LNPs of the BBG/p-CMV-GFP complex. Mannose receptor-facilitated in vivo DC-targeted vaccination (s.c.) with the electrostatic complex of LNPs of DSG/pCMV-MART1 stimulated long-lasting (270 days post B16F10 tumor challenge) antimelanoma immunity under prophylactic conditions. Remarkably, under therapeutic settings, vaccination (s.c.) with LNPs of the DSG/pCMV-MART1 complex significantly delayed melanoma growth and improved the survival of mice with melanoma. These findings demonstrate that this nonviral delivery system offers a resilient and potential approach to deliver DNA vaccines encoding tumor antigens to DCs in vivo with high efficacy.

Comparison of Protective Potency of DNA and Live Vaccines Expressing A2-CPA-CPB-CTE Antigens against Visceral Leishmaniasis in Syrian Hamster as Preliminary Study.

BACKGROUND: Visceral leishmaniasis is the most severe form of leishmaniasis caused by Leishmania (L.) donovani complex. Drug-resistant strains have been developed as a consequence of the current chemotherapeutic interventions, which has increased the need for advanced preventive and therapeutic strategies. A2-CPA-CPB-CTE-recombinant strain of L. tarentolae, which is non-pathogenic to humans, was shown protective in live vaccine as well as its DNA vaccine counterpart in both murine and canine models. METHODS: We evaluated the effectiveness of these DNA and live vaccination harboring A2-CPA-CPB-CTE in protecting hamsters against L. infantum infection using prime-boost regimens, namely DNA/DNA and Live/Live (n=9 hamsters per group). Cationic solid lipid nanoparticles (cSLN) were utilized as an adjuvant for DNA priming and electroporation for boosting DNA. At different time points post-challenge, parasite burden and body weight as well as humoral immune responses were measured. RESULTS: Both immunization strategies partially protect hamsters against L. infantum challenge. This protective immunity is associated with remarkable decrease in parasite load in liver and spleen of vaccinated hamsters eight weeks after challenge compared to control group. CONCLUSION: Both test groups (DNA/DNA and Live/Live) elicited high levels of IgG2 and total IgG as humoral immune responses and lower level of parasite propagation in both liver and spleen.

Plant-Derived Natural Compounds in Genetic Vaccination and Therapy for HPV-Associated Cancers.

Antigen-specific immunotherapy and, in particular, DNA vaccination provides an established approach for tackling human papillomavirus (HPV) cancers at different stages. DNA vaccines are stable and have a cost-effective production. Their intrinsic low immunogenicity has been improved by several strategies with some success, including fusion of HPV antigens with plant gene sequences. Another approach for the control of HPV cancers is the use of natural immunomodulatory agents like those derived from plants, that are able to interfere in carcinogenesis by modulating many different cellular pathways and, in some instances, to reduce chemo- and radiotherapy resistance of tumors. Indeed, plant-derived compounds represent, in many cases, an abundantly available, cost-effective source of molecules that can be either harvested directly in nature or obtained from plant cell cultures. In this review, an overview of the most relevant data reported in literature on the use of plant natural compounds and genetic vaccines that include plant-derived sequences against HPV tumors is provided. The purpose is also to highlight the still under-explored potential of multimodal treatments implying DNA vaccination along with plant-derived agents.

Integrated transcriptomic and quantitative proteomic analysis identifies potential RNA sensors that respond to the Ag85A DNA vaccine.

OBJECTIVE: DNA vaccine has emerged as a promising approach with potential for Tuberculosis (TB) prevention in adults. However, the mechanism behind DNA vaccines is still largely unknown. MATERIALS AND METHODS: Utilizing the CRISPR/Cas9 technique, we engineered Ag85A mutated dendritic cells (Ag85A-M-DCs) in which the Ag85A mRNA derived from Mycobacterium tuberculosis was expressed but not the corresponding protein. Control cells (Ag85A-DCs) expressed both Ag85A mRNA and protein. To better understand the mechanism of antigen presentation following DNA vaccination, integrated transcriptomic and proteomic analysis of dendritic cells (DCs), Ag85A-DCs, and Ag85A-M-DCs were performed. RESULTS: A total of 723, 278, and 933 differentially expressed genes (DEGs), and 209, 134, and 509 differentially expressed proteins (DEPs) were identified between Ag85A-M-DCs and DCs, Ag85A-DCs and DCs, and Ag85A-M-DCs and Ag85A-DCs, respectively. Integration analysis detected 59, 15, and 64 associated DEGs/DEPs with the same expression trend between Ag85A-M-DCs and DCs, Ag85A-DCs and DCs, and Ag85A-M-DCs and Ag85A-DCs, respectively. KEGG pathway analysis showed that chemokine signaling pathway and MAPK signaling pathway were enriched in all three pairs of comparisons. The protein and protein interaction network revealed that ANXA1 was in the top 10 high-degree hub genes closely related to other genes in all three pairs of comparisons. CONCLUSION: The results indicated that Ag85A DNA vaccine might transmit immunogenicity information and induce immune responses by activating chemokine signaling pathway and MAPK signaling pathway. ANXA1 may serve as a key target molecule of the Ag85A vaccine with additional potential for TB prevention.

Intranasal inoculations of naked or PLGA-PEI nanovectored DNA vaccine induce systemic and mucosal antibodies in pigs: A feasibility study.

Mucosa are the routes of entry of most pathogens into animals' organisms. Reducing the important global burden of mucosal infectious diseases in livestock animals is required in the field of veterinary public health. For veterinary respiratory pathogens, one possible strategy is the development of intranasal (IN) DNA vaccination. The aim of this study was to assess the feasibility of IN DNA vaccination in pigs, an important species in livestock production industry, and a source of zoonotic diseases. To achieve this goal, we used a DNA vaccine against pseudorabies virus (PrV) encoding the immunogenic glycoprotein B (pcDNA3-gB plasmid). When pigs were inoculated with the naked DNA vaccine through the IN route, PrV-specific IgG and IgA type antibodies were detected in porcine sera. Interestingly, mucosal salivary IgA antibodies against PrV were also detected, at similar levels to those measured following intramuscular injection (positive controls). Furthermore, the IN delivery of pcDNA3-gB combined with PLGA-PEI nanoparticles resulted in similar levels of antibodies but was associated with an increase in the duration of detection of mucosal IgA for 2 out of 3 pigs. Our results suggest that there is room to improve the efficacy of IN DNA vaccination in pigs through optimization of IN inoculations, for example by using nanoparticles such as PLGA-PEI. Further studies will be dedicated to optimizing and testing the protective potential of IN DNA vaccination procedures against PrV.