DNA Vaccine In Mice Research Articles

Tolerability of a piezoelectric microneedle electroporator in human subjects.

Electroporation, or the use of electric pulses to facilitate the intracellular delivery of DNA, RNA, and other molecules, is a well-established technique, that has been demonstrated to significantly augment the immunogenicity of DNA/mRNA vaccines and therapeutics. However, the clinical translation of traditional electroporators has been limited due to high costs, large size, complex user operation, and poor tolerability in humans due to nerve stimulation. In prior work, we introduced ePatch: an ultra-low-cost, handheld, battery-free electroporator employing a piezoelectric pulser coupled with a microneedle electrode array that showed enhanced immunogenic responses to an intradermal SARS-CoV-2 DNA vaccine in mice. The current study shifts focus from efficacy to tolerability, hypothesizing that ePatch's microneedle array, which localizes the electric field to the superficial skin strata, will minimize nerve stimulation and improve patient comfort. We tested this hypothesis in 14 healthy adults, monitoring pain and other potential adverse effects associated with electroporation. Compared to the insertion of a traditional hypodermic needle, the ePatch was less painful. Adverse effects such as pain, tenderness, erythema and swelling at the application sites were minimal, transient, and statistically indistinguishable between the experimental and placebo ePatch application, suggesting excellent tolerability towards electroporation. In summary, ePatch has a favorable tolerability profile in humans and offers the potential for the safe use of electroporation in a variety of clinical settings, including DNA and mRNA vaccination.

Effects of single and multiple bouts of eccentric exercise on immune responses to HSV-gD1 DNA vaccination in mice

Abstract DNA vaccines comprise one modality of novel vaccination techniques. DNA vaccines employ intramuscular injection of DNA that codes for specific antigens from a pathogen. Prolonged eccentric endurance exercise can stimulate immune responses, possibly by increasing inflammatory mediators. This study aimed to determine the effects of one or three sessions of downhill running on the effectiveness of Herpes simplex virus type I glycoprotein D (HSV-gD1) DNA vaccination. Mice without or with one and three sessions of downhill running were vaccinated intramuscularly with the DNA vaccine or the empty vector. Cellular immune responses were assessed using an in vitro assay of lymphocyte proliferation in response to recall antigens. Also, a virus neutralisation test was used to determine the levels of neutralising antibodies in serum samples. In addition, Th1 and Th2 immune responses, and the ratio of the Th1/Th2 is considered an indicator of immune responses to infection in the current study. Multiple bouts of downhill running increased humoral immune responses after immunisation, also although Th2 immune responses decreased, lymphocyte proliferation and Th1/Th2 ratio immune responses increased following the viral challenge compared to the other groups. In addition, the timing of the 3-session exercise regimen plus DNA vaccination immediately following the last exercise session, as opposed to DNA vaccination 48 h before a single session of exercise, may account for the greater efficacy of DNA vaccination in the multiple bouts of the intensive exercise group. These findings indicate that short-term repeated bouts of eccentric endurance exercise have increased immune responses to intramuscular DNA-based vaccines.

Effect of plasmid size on the immune response to Japanese Encephalitis virus DNA vaccine in mice

Background: The envelope (E) protein, along with the pre-membrane (prM) protein of the Japanese encephalitis virus (JEV), are importantmediators of humoral immunity against the virus. Objective: To establish whether the plasmid size could influence the immunogenicityof this DNA vaccine candidate in terms of antibody inductive capacity. Methods: The prM and E genes of JEV were cloned in pCEP4 andpCDNA to produce the constructs pMEa and pcMEa, respectively. The former construct was approximately double the size of the latter.The plasmid constructs were used to immunize mice, both in terms of equal weight, as well as equal number of molecules. Results:The antibody-inductive capacity (both anti-JEV and neutralizing antibodies) of pCDNA was superior to that of the pCEP4. Importantly,the mice challenge studies indicated that the higher titer of neutralizing antibodies of the pCDNA group lead to higher survival in thisgroup. Conclusion: A smaller plasmid would be better for preparing a DNA vaccine construct.

The ultrasonically treated nanoliposomes containing PCV2 DNA vaccine expressing gC1qR binding site mutant Cap is efficient in mice.

Nowadays, vaccines are broadly used to prevent porcine circovirus type 2 (PCV2) infection-induced expenditures, but the virus is still spreading among pigs. The current PCV2 vaccines all rely on the immunogenicity of Cap, yet our previous studies found that Cap is also the major component mediating the PCV2 infection-induced immune suppression through its interaction with host gC1qR. Thereby, new vaccines are still necessary for PCV2 prevention and control. In this study, we constructed a new PCV2 DNA vaccine expressing the gC1qR binding site mutant Cap. We introduced the Intron A and WPRE elements into the vector to improve the Cap expression level, and fused the IL-2 secretory signal peptides to the N-terminal of Cap to mediate the secretion of Cap. We also screened and selected chemokines CXCL12, CCL22, and CCL25 to migrate dendritic cells. In addition, we contained the vectors with PEI and then ultrasonic them into nano size to enhance the entrance of the vectors. Finally, the animal experiments showed that the new PCV2 DNA vaccine expressing the gC1qR binding site mutant Cap could induce stronger humoral and cellular immune responses than the PCV2 DNA vaccine expressing the wild-type Cap and the non-ultrasonic treated PCV2 DNA vaccine in mice, and protect the mice from PCV2 infection and lung lesions. The results indicate the new PCV2 DNA vaccine expressing the gC1qR binding site mutant Cap has a certain development value, and provide new insight into the development of novel PCV2 vaccines.

Co-administration of a plasmid encoding CD40 or CD63 enhances the immune responses to a DNA vaccine against bovine viral diarrhea virus in mice.

Bovine viral diarrhea virus (BVDV) causes substantial economic losses in the livestock industry worldwide. Plasmids encoding the BVDV E2 protein are potential DNA vaccines against BVDV, but their immunogenicity has been insufficient. Here, we investigated the adjuvant effect of CD40 and CD63 plasmids on the immune responses to a BVDV E2 DNA vaccine in mice. We constructed pUMVC4a-based plasmids encoding the BVDV E2 protein (pE2), mouse CD40 (pCD40), or mouse CD63 (pCD63). Protein expression by each plasmid was confirmed through Western blot analysis and immunofluorescence staining of cultured cell lines. BALB/c mice were immunized intradermally twice with pE2 in combination with, or without, pCD40 or pCD63, with 3 weeks between the two doses. pE2 with pCD40 induced significantly higher neutralizing antibody titers against BVDV than pE2 alone. pE2 with pCD63 induced significantly higher anti-E2 IgG2a antibody titers than pE2 alone. Furthermore, pE2 with pCD40 or pCD63 induced significantly increased lymphocyte proliferation and interferon (IFN)-γ production in response to BVDV, compared with E2 alone. These results suggest that a plasmid encoding CD40 or CD63 can be used as an adjuvant to enhance immune responses to DNA vaccines against BVDV.

Invaplex functions as an intranasal adjuvant for subunit and DNA vaccines co-delivered in the nasal cavity of nonhuman primates

Development of intranasal vaccines for HIV-1 and other mucosal pathogens has been hampered by the lack of adjuvants that can be given safely to humans. We have found that an intranasal Shigella vaccine (Invaplex) which is well tolerated in humans can also function as an adjuvant for intranasal protein and DNA vaccines in mice. To determine whether Invaplex could potentially adjuvant similar vaccines in humans, we simultaneously administered a simian immunodeficiency virus (SIV) envelope (Env) protein and DNA encoding simian-human immunodeficiency virus (SHIV) with or without Invaplex in the nasal cavity of female rhesus macaques. Animals were intranasally boosted with adenoviral vectors expressing SIV env or gag,pol to evaluate memory responses. Anti-SIV antibodies in sera and nasal, genital tract and rectal secretions were quantitated by ELISA. Intracellular cytokine staining was used to measure Th1-type T cells in blood. Macaques given DNA/protein immunizations with 0.5 mg Invaplex developed greater serum IgG, nasal IgA and cervicovaginal IgA responses to SIV Env and SHIV Gag,Pol proteins when compared to non-adjuvanted controls. Rectal IgA responses to Env were only briefly elevated and not observed to Gag,Pol. Invaplex increased frequencies of IFNγ-producing CD4 and CD8 T cells to the Env protein, but not T cell responses induced by the DNA. Ad-SIV boosting increased Env-specific polyfunctional T cells and Env- and Gag,Pol-specific antibodies in serum and all secretions. The data suggest that Invaplex could be highly effective as an adjuvant for intranasal protein vaccines in humans, especially those intended to prevent infections in the genital or respiratory tract.

1217P - Anti-CSPG4 DNA vaccination as a promising strategy for the treatment of CSPG4+ tumours: A comparative oncology trial

BackgroundAmong the most interesting targets for immunotherapeutic approaches, the Chondroitin Sulfate Proteoglycan (CSPG)4 stands out, with low expression in healthy tissues, high expression in several solid tumors and a key role in cancer progression. Because of the translational power of dogs as pre-clinical models for human malignancies and the CSPG4 over-expression by both human and canine malignant melanoma (MM), we demonstrated the safety and the clinical effectiveness of a xenogeneic human (Hu)-CSPG4 DNA vaccine in client-owned canine patients with stage II-III surgically resected CSPG4+ MM. However, Hu-CSPG4 vaccine was barely effective in activating human T cells from healthy donors in vitro. Based on these results, we aimed to increase the translational power of our approach and to extend it for the treatment of CSPG4+ tumors other than MM. MethodsWe generated a hybrid plasmid, derived in part from the Hu- and in part from the dog (Do)-CSPG4 sequences (HuDo-CSPG4). We tested the safety, immunogenicity and anti-tumor potential of HuDo-CSPG4 DNA vaccine in mice, in dogs with stage II-IV surgically resected CSPG4+ MM and in a human setting in vitro. We evaluated the potential of CSPG4-immune-targeting also for osteosarcoma (OSA). ResultsChimeric HuDo-CSPG4 vaccination is strongly immunogenic in mice. In canine patients, the procedure is safe and induces antibodies (Ab) against both Hu- and Do-CSPG4, with a higher affinity and anti-tumor potential as compared to Hu-CSPG4. Clinically, HuDo-CSPG4 is effective in increasing the overall survival of vaccinated canine MM patients as compared to controls. Data obtained in vitro with T cells from human healthy donors suggest HuDo-CSPG4 is more immunogenic than Hu-CSPG4. Moreover, CSPG4 over-expression was demonstrated in human and canine OSA. Interestingly, anti-CSPG4 specific monoclonal Ab and sera derived from CSPG4 vaccinated canine MM patients showed to significantly inhibit human and canine CSPG4+ OSA cell proliferation, migration and osteospheres generation. ConclusionsThese results provide the rationale to propose HuDo-CSPG4 vaccination for the treatment of canine CSPG4+ tumors, to be successfully translated in a human setting. Legal entity responsible for the studyFederica Cavallo. FundingAssociazione Italiana per la Ricerca sul Cancro (AIRC), Italian Ministry of Health. DisclosureAll authors have declared no conflicts of interest.

MOMP and MIP DNA-loaded bacterial ghosts reduce the severity of lung lesions in mice after Chlamydia psittaci respiratory tract infection.

Chlamydia psittaciis a well known zoonotic pathogen that can lead to severe respiratory disease in poultry, pet birds and humans. Development of an effective and safe vaccine would be the most effective way to control C. psittaci infection. In this study, we used bacterial ghosts (BGs) as a delivery vehicle to evaluate the protective effects of major outer membrane protein (MOMP) and macrophage infectivity potentiator (MIP) DNA vaccines in mice. We found that MOMP/MIP DNA-loaded BGs elicited a better immune response than a naked DNA vaccine, giving increased IgG titers, lymphocyte proliferation responses and higher levels of IFN-γ. After challenge infection, MOMP/MIP DNA-loaded BGs-immunized mice showed lower chlamydial load and inflammation pathology in lung tissues. In addition, we found that MOMP and MIP co-immunization or a heterologous prime-boost strategy could induce stronger immune responses and better protective efficacy against C. psittaci infection. Together, the above results suggest that BGs can act as an effective delivery vehicle for C. psittaci DNA vaccines, and co-immunization or heterologous prime-boost strategy can enhance protective efficacy against infection, thereby providing an alternative strategy for the design of vaccines against C. psittaci.

Significant body mass increase by oral administration of a cascade of shIL21-MSTN yeast-based DNA vaccine in mice

Base on the practical of MSTN-specific yeast-based protein vaccine in mice as described previously, this research was designed for developing a better DNA vaccine (a cascade of shIL21-MSTN yeast-based DNA vaccine) than solely MSTN yeast-based DNA vaccine to block the endogenous MSTN in the murine model. We first constructed the target vectors, including CMV-driven MSTN expression vector and a combined shIL21-MSTN vector which containing MSTN expression cassette and shIL21 (short hairpin RNA-IL21) expression cassette. After necessary validation, recombinant yeast vaccines harboring different vectors were well prepared. Subsequently, after 2-month administration, the MSTN-specific immune response was detected with western blots. The commercial ELISA assays indicated that the production of IL21 and IL6 were decreased compared with control groups. More importantly, the MSTN-specific antibody titer was much higher in the shIL21-MSTN group than MSTN group, which was consistent with the western blots result. The most important finding was significant body mass increased after oral administration of these yeast-based DNA vaccines, in which the shIL21-MSTN vaccine is slightly higher than the sole MSTN vaccine in mice. In this study, we confirmed the role of different MSTN-specific yeast-based DNA vaccines on increasing body mass in mice, to provide a good inspiration for livestock breeding through the new type of immunoregulatory method. On the other hand, we also detected the possible modulating role of shIL21 on the dendritic cell-mediated immune function which needs more practical application and deeper exploration.

Co-expression of IL-15 enhances anti-neuroblastoma effectivity of a tyrosine hydroxylase-directed DNA vaccination in mice

Long-term survival of high-risk neuroblastoma (NB) patients still remains under 50%. Here, we report the generation, in vitro characterization and anti-tumor effectivity of a new bicistronic xenogenic DNA vaccine encoding tyrosine hydroxylase (TH) that is highly expressed in NB tumors, and the immune stimulating cytokine interleukin 15 (IL-15) that induces cytotoxic but not regulatory T cells. The DNA sequences of TH linked to ubiquitin and of IL-15 were integrated into the bicistronic expression vector pIRES. Successful production and bioactivity of the vaccine-derived IL-15- and TH protein were shown by ELISA, bioactivity assay and western blot analysis. Further, DNA vaccine-driven gene transfer to the antigen presenting cells of Peyer’s patches using attenuated Salmonella typhimurium that served as oral delivery system was shown by immunofluorescence analysis. The anti-tumor effect of the generated vaccine was evaluated in a syngeneic mouse model (A/J mice, n = 12) after immunization with S. typhimurium (3× prior and 3× after tumor implantation). Importantly, TH-/IL-15-based DNA vaccination resulted in an enhanced tumor remission in 45.5% of mice compared to controls (TH (16.7%), IL-15 (0%)) and reduced spontaneous metastasis (30.0%) compared to controls (TH (63.6%), IL-15 (70.0%)). Interestingly, similar levels of tumor infiltrating CD8+ T cells were observed among all experimental groups. Finally, co-expression of IL-15 did not result in elevated regulatory T cell levels in tumor environment measured by flow cytometry. In conclusion, co-expression of the stimulatory cytokine IL-15 enhanced the NB-specific anti-tumor effectivity of a TH-directed vaccination in mice and may provide a novel immunological approach for NB patients.

Immunotherapeutic effects of Mycobacterium tuberculosis rv3407 DNA vaccine in mice

Tuberculosis (TB) is a major global public health problem. Latent TB infection (LTBI) is a major source of active TB. New vaccines to treat LTBI are urgently demanded. In this study, the gene encoding latency-associated antigen Rv3407 of Mycobacterium tuberculosis (MTB) rv3407 DNA vaccine was used to prepare and the immunogenicity and therapeutic effects were evaluated. Normal mice were immunized intramuscularly three times at two-week intervals with sterile water for injection, plasmid vector pVAX1, M. vaccae vaccine, ag85a DNA or rv3407 DNA. TB-infected mice were immunized intramuscularly three times at two-week intervals with phosphate-buffered saline (PBS) and rv3407 DNA. The normal mice immunized with rv3407 DNA or ag85a DNA showed higher levels of interferon-gamma (IFN-γ) in stimulated spleen lymphocyte culture supernatants, and had more Th1 cells and an elevated ratio of Th1/Th2 immune cells in whole blood, indicating that a Th1-type immune response was predominant. The levels of anti-Ag85A or anti-Rv3407 IgG antibody were significantly increased in the ag85a DNA and rv3407 DNA groups compared to the sterile water for injection, vector, and M. vaccae groups (p < .0001). Compared with the PBS group, the rv3407 DNA group had pulmonary bacterial loads that were lower by 0.56 log10 (p < .01). The mice vaccinated with rv3407 DNA developed antigen-specific cellular and humoral responses. The rv3407 DNA is a potential DNA vaccine candidate against TB.

Immunogenicity and therapeutic effects of a Mycobacterium tuberculosis rv2190c DNA vaccine in mice

BackgroundTuberculosis (TB) is a major global public health problem. New treatment methods on TB are urgently demanded. In this study, Mycobacterium tuberculosis (MTB) rv2190c DNA vaccine was prepared and its immunogenicity and therapeutic effects were evaluated.ResultsNon-infected mice immunized with rv2190c DNA or ag85a DNA showed higher levels of interferon-gamma (IFN-γ) in stimulated spleen lymphocyte culture supernatants, and had more Th1 cells and an elevatory ratio of Th1/Th2 immune cells in whole blood, indicating that Th1-type immune response was predominant. Compared with the saline group, ag85a DNA group and rv2190c DNA group in the infected mice decreased the lung colony-forming units (CFUs) by 0.533 and 0.283 log10, and spleen CFUs by 0.425 and 0.321 log10 respectively, and pathological lesion.ConclusionsThe rv2190c DNA had some immunotherapeutic effect on TB.

Humoral and Cellular Immunogenicity Induced by Avian Influenza A (H7N9) DNA Vaccine in Mice.

BackgroundIn March 2013, human infection with avian influenza A (H7N9) virus emerged in China, causing serious public health concerns and raising the possibility of avian-source pandemic influenza. Thus, the development of an effective vaccine for preventing and rapidly controlling avian influenza A (H7N9) virus is needed. In this study, we evaluated the immunogenicity of a synthetic DNA vaccine against H7 HA antigens in mice.Materials and MethodsThe synthetic consensus H7 HA DNA vaccine (25 or 50 µg) was administered to BALB/c mice at 0, 14, and 28 days by intramuscular injection followed by electroporation. Humoral and cellular immune responses were analyzed in a hemagglutination inhibition test and interferon-gamma enzyme-linked immunospot (ELISpot) assay, respectively.ResultsH7 HA-vaccinated mice showed 100% seroprotection and seroconversion rate against H7N9 reassortant influenza virus after both second and third immunizations. The geometric mean titer by the hemagglutination inhibition test increased with an increasing number of immunizations. However, there was no significant difference in geometric titer between the two groups injected with 25 and 50 µg of H7 HA DNA vaccine after two (79.98 vs. 107.65, P = 0.39) and three (159.96 vs. 215.28, P = 0.18) doses. In addition, the ELISpot assay revealed that administration of H7 HA DNA vaccine induced potent interferon-gamma production from mouse splenocytes.ConclusionsThis study demonstrated the humoral and cellular immunogenicity of synthetic consensus H7 HA DNA vaccine in mice. This work demonstrates the potential of the H7 HA DNA vaccine as an efficient tool for the rapid control of emerging influenza A (H7N9) virus.

Antigen Targeting to Human HLA Class II Molecules Increases Efficacy of DNA Vaccination.

It has been difficult to translate promising results from DNA vaccination in mice to larger animals and humans. Previously, DNA vaccines encoding proteins that target Ag to MHC class II (MHC-II) molecules on APCs have been shown to induce rapid, enhanced, and long-lasting Ag-specific Ab titers in mice. In this study, we describe two novel DNA vaccines that as proteins target HLA class II (HLA-II) molecules. These vaccine proteins cross-react with MHC-II molecules in several species of larger mammals. When tested in ferrets and pigs, a single DNA delivery with low doses of the HLA-II-targeted vaccines resulted in rapid and increased Ab responses. Importantly, painless intradermal jet delivery of DNA was as effective as delivery by needle injection followed by electroporation. As an indication that the vaccines could also be useful for human application, HLA-II-targeted vaccine proteins were found to increase human CD4+ T cell responses by a factor of ×103 in vitro. Thus, targeting of Ag to MHC-II molecules may represent an attractive strategy for increasing efficacy of DNA vaccines in larger animals and humans.

A DNA vaccine encoding VP22 of herpes simplex virus type I (HSV-1) and OprF confers enhanced protection from Pseudomonas aeruginosa in mice

Pseudomonas aeruginosa antimicrobial resistance is a major therapeutic challenge. DNA vaccination is an attractive approach for antigen-specific immunotherapy against P. aeruginosa. We explored the feasibility of employing Herpes simplex virus type 1 tegument protein, VP22, as a molecular tool to enhance the immunogenicity of an OprF DNA vaccine against P. aeruginosa. Recombinant DNA vaccines, pVAX1-OprF, pVAX1-OprF-VP22 (encoding a n-OprF-VP22-c fusion protein) and pVAX1-VP22-OprF (encoding a n-VP22-OprF-c fusion protein) were constructed. The humoral and cellular immune responses and immune protective effects of these DNA vaccines in mice were evaluated. In this report, we showed that vaccination with pVAX1-OprF-VP22 induced higher levels of IgG titer, T cell proliferation rate. It also provided better immune protection against the P. aeruginosa challenge when compared to that induced by pVAX1-OprF or pVAX1-VP22-OprF DNA vaccines. Molecular mechanistic analyses indicated vaccination with pVAX1-OprF-VP22 triggered immune responses characterized by a preferential increase in antigen specific IgG2a and IFN-γ in mice, indicating Th1 polarization. We concluded that VP22 is a potent stimulatory molecular tool for DNA vaccination when fused to the carboxyl end of OprF gene. Our study provides a novel strategy for prevention and treatment of P. aeruginosa infection.

Humoral and cellular immune responses to modified hepatitis B plasmid DNA vaccine in mice

Purpose : To evaluate the immunogenicity and types of immune response of a quality-controlled modified recombinant hepatitis B surface antigen (HBsAg) plasmid encoding HBsAg in mice. Methods : The characterized plasmid DNA was used in the immunization of Balb/c mice. Three groups of mice were intramuscularly injected with three different concentrations (50, 25 and 10 μg/100 μL) of the modified plasmid. Humoral immune response was monitored by enzyme-linked immunosorbent assay (ELISA), while cellular immune response was investigated by analysis of spleen cytokine profile (TNFα, IFN γ and IL2) as well as CD69 expression level in CD4 and CD8 positive cells. Results : In general, the activated CD4 cells showing intracellular cytokines were higher than CD8 positive population of cells (p < 0.05). These findings indicate that the vaccine induced both a humoral and cellular immunity. Cytokine profile also showed high levels of TNFα, IFN γ and IL2 and CD69 expression in the group of animals immunized at a dose of 10 μg when compared to control group (p < 0.05). Conclusion : A 10 μg dose intramuscular injection of the modified DNA-based vaccine encoding HBsAg in mice induces both high humoral and cellular immune responses. Keywords : Hepatitis B virus, Plasmid DNA, Vaccine, Spleen cytokines, Humoral and cellular immune responses

Antitumor vaccination of prostate cancer patients elicits PD-1/PD-L1 regulated antigen-specific immune responses

ABSTRACTWe have previously reported that tumor antigen-specific DNA vaccination in mice led to an increase in IFNγ-secreting T cells and an increase in tumor expression of PD-L1. Further, we demonstrated that increasing the encoded antigen's MHC-binding affinity led to increased PD-1 expression on antigen-specific CD8+ T cells. Together these phenomena provided resistance to antitumor immunization that was abrogated with PD-1/PD-L1 blockade. We consequently sought to determine whether similar regulation occurred in human patients following antitumor immunization. Using clinical samples from prostate cancer patients who were previously immunized with a DNA vaccine, we analyzed changes in checkpoint receptor expression on antigen-specific CD8+ T cells, the effect of PD-1 blockade on elicited immune responses, and for changes in checkpoint ligand expression on patients' circulating tumor cells (CTCs). We observed no significant changes in T-cell expression of PD-1 or other checkpoint receptors, but antigen-specific immune responses were detected and/or augmented with PD-1 blockade as detected by IFNγ and granzyme B secretion or trans vivo DTH testing. Moreover, PD-L1 expression was increased on CTCs following vaccination, and this PD-L1 upregulation was associated with the development of sustained T-cell immunity and longer progression-free survival. Finally, similar results were observed with patients treated with sipuleucel-T, another vaccine targeting the same prostate antigen. These findings provide in-human rationale for combining anticancer vaccines with PD-1 blocking antibodies, particularly for the treatment of prostate cancer, a disease for which vaccines have demonstrated benefit and yet PD-1 inhibitors have shown little clinical benefit to date as monotherapies.

Immunogenicity and safety of xenogeneic vascular endothelial growth factor receptor-2 DNA vaccination in mice and dogs.

Vascular endothelial growth factor receptor-2 (VEGFR-2) is an attractive target in oncology due to its crucial role in angiogenesis. In this study a DNA vaccine coding for human VEGFR-2 was evaluated in healthy mice and dogs, administered by intradermal injection and electroporation. In mice, three doses and vaccination schedules were evaluated. Cellular immune responses were measured by intracellular IFN-gamma staining and a cytotoxicity assay and antibodies by ELISA. Safety was assessed by measuring regulatory T cells and myeloid derived suppressor cells and a wound healing assay. The vaccine was subsequently evaluated in dogs, which were vaccinated three times with 100μg. Cellular immune responses were measured by intracellular IFN-gamma staining and antibodies by a flow cytometric assay. In mice, maximal cellular responses were observed after two vaccinations with 5μg. Humoral responses continued to increase with higher dose and number of vaccinations. No abnormalities in the measured safety parameters were observed. The vaccine was also capable of eliciting a cellular and humoral immune response in dogs. No adverse effects were observed, but tolerability of the electroporation was poor. This study will facilitate the evaluation of the vaccine in tumor bearing animals, ranging from rodent models to dogs with spontaneous tumors.

A novel dendritic-cell-targeting DNA vaccine for hepatitis B induces T cell and humoral immune responses and potentiates the antivirus activity in HBV transgenic mice

Strategies for inducing an effective immune response following vaccination have focused on targeting antigens to dendritic cells (DCs) through the DC-specific surface molecule DEC-205. The immunogenicity and efficacy of DNA vaccination can also be enhanced by fusing the encoded antigen to single-chain antibodies directed against DEC-205. Here, we investigated this promising approach for its enhancement of hepatitis B virus (HBV)-specific cellular and humoral immune responses and its antiviral effects in HBV transgenic mice. A plasmid DNA vaccine encoding mouse DEC-205 single-chain fragment variable (mDEC-205-scFv) linked with the hepatitis B surface antigen (HBsAg) was constructed. Vaccination with this fusion DNA vaccine in HBV transgenic mice induced robust antiviral T cell and antibody immunity against HBsAg. The levels of serum-circulating HBsAg and the HBV DNA copy number were downregulated by the induction of a higher HBsAg-specific response. Thus, in this study, we demonstrated the therapeutic efficacy of the novel mDEC-205-scFv-fused DNA vaccine in a mouse model of immune-tolerant, chronic HBV infection.

Adjuvants may reduce in vivo transfection levels for DNA vaccination in mice leading to reduced antigen-specific CD8+ T cell responses

Adjuvants for DNA vaccination are designed to promote transformation of transgenes into target cells and increase inflammation in the site of injection, with resultant immune cell recruitment. Numerous studies indicated cationic liposomes as effective adjuvants for DNA vaccination due to their ability to promote in vivo transfection and innate immune system activation. Commercial reagents as Adjuplex and in vivo-JetPEI are also intended to facilitate DNA vaccination. Here, we evaluate the adjuvant properties of cationic liposomes, Adjuplex and in vivo-JetPEI compared to injection of DNA without adjuvant. In mice vaccinated with piggyBac pDNA vaccines, we assessed in vivo antigen expression, innate immune responses in draining lymph nodes, and antigen-specific T cell responses in spleens and blood. Surprisingly, vaccination with DNA in PBS emerged as the most efficient in promoting in vivo transfection and consequent antigen expression, while the addition of adjuvant reduced the amount of antigen expressed. On the other hand, we discovered higher numbers of innate immune cells and activated dendritic cells in the lymph nodes of mice injected with adjuvants than those vaccinated in PBS. The analysis of eGFP-specific immune responses revealed that all the different immunizations induced functional antigen-specific T cells in spleens, although only T cells generated by non-adjuvant vaccination and Adjuplex were identified in the blood of vaccinated mice. These results provide insight into the effects of these 3 adjuvants and may facilitate appropriate use off adjuvants by researchers using DNA vaccines in laboratory animals.