Tetravalent DNA Research Articles

Novel Complex of PD-L1 Aptamer and Holliday Junction Enhances Antitumor Efficacy in Vivo.

Blocking the PD-1/PD-L1 pathway can diminish immunosuppression and enhance anticancer immunity. PD-1/PD-L1 blockade can be realized by aptamers, which have good biocompatibility and can be synthesized in quantity economically. For in vivo applications, aptamers need to evade renal clearance and nuclease digestion. Here we investigated whether DNA nanostructures could be used to enhance the function of PD-L1 aptamers. Four PD-L1 aptamers (Apt) were built into a Holliday Junction (HJ) to form a tetravalent DNA nanostructure (Apt-HJ). The average size of Apt-HJ was 13.22 nm, which was above the threshold for renal clearance. Apt-HJ also underwent partial phosphorothioate modification and had improved nuclease resistance. Compared with the monovalent PD-L1 aptamer, the tetravalent Apt-HJ had stronger affinity to CT26 colon cancer cells. Moreover, Apt-HJ markedly boosted the antitumor efficacy in vivo vs. free PD-L1 aptamers without raising systemic toxicity. The results indicate that multiple aptamers attached to a DNA nanostructure may significantly improve the function of PD-L1 aptamers in vivo.

Tetravalent dengue DNA vaccine is not immunogenic when delivered by retrograde infusion into salivary glands

Introduction and backgroundA tetravalent DNA vaccine for Dengue virus is under development but has not yet achieved optimal immunogenicity. Salivary glands vaccination has been reported efficacious in rodents and dogs. We report on a pilot study testing the salivary gland as a platform for a Dengue DNA vaccine in a non-human primate model.Materials and methodsFour cynomolgus macaques were used in this study. Each macaque was pre-medicated with atropine and sedated with ketamine. Stensen’s duct papilla was cannulated with a P10 polyethylene tube, linked to a 500ul syringe. On the first two infusions, all macaques were infused with 300ul of TVDV mixed with 2 mg of zinc. For the 3rd infusion, to increase transfection into salivary tissue, two animals received 100uL TVDV mixed with 400uL polyethylenimine 1μg/ml (PEI) and the other two animals received 500uL TVDV with zinc. Antibody titers were assessed 4 weeks following the second and third infusion.Results and conclusionsSGRI through Stensen’s duct is a well-tolerated, simple and easy to reproduce procedure. TVDV infused into macaques salivary glands elicited a significantly weaker antibody response than with different delivery methods.

Long-Term Protection Elicited by a DNA Vaccine Candidate Expressing the prM-E Antigen of Dengue Virus Serotype 3 in Mice

Dengue virus (DENV) is the causative agent of dengue, and its incidence has increased 30-fold in the past five decades. Among the four cocirculating serotypes, DENV3 is associated with an increased number of severe infections and has become widespread. Vaccination is the mainstay of prevention in reducing disease burden. Previously, the protective efficacy of DNA vaccine candidates toward DENV1, 2, and 4 was confirmed in mice. In this study, a DNA vaccine candidate (pVAX1-D3ME) expressing the prM and E proteins of DENV3 was constructed, and then the immunogenicity and protection were assessed in mice to further develop a tetravalent dengue vaccine. Moreover, the cross-reactive immune responses against the other three serotypes were investigated. The results showed that three doses of 50 μg of pVAX1-D3ME were sufficient to induce strong antigen-specific T cell responses and robust and consistent neutralizing antibodies. Additionally, immunization with pVAX1-D3ME offered protective immunity against not only DENV3 but also the other three serotypes, which could be observed even after 12 months. This study shows great promise for the further evaluation of a dengue tetravalent DNA vaccine candidate in large animal models, including non-human primates.

Enhanced immunogenicity and protective efficacy of a tetravalent dengue DNA vaccine using electroporation and intradermal delivery

Phase 1 clinical trials with a DNA vaccine for dengue demonstrated that the vaccine is safe and well tolerated, however it produced less than optimal humoral immune responses. To determine if the immunogenicity of the tetravalent dengue DNA vaccine could be enhanced, we explored alternate, yet to be tested, methods of vaccine administration in non-human primates. Animals were vaccinated on days 0, 28 and 91 with either a low (1 mg) or high (5 mg) dose of vaccine by the intradermal or intramuscular route, using either needle-free injection or electroporation devices. Neutralizing antibody, IFN-γ T cell and memory B cell responses were compared to a high dose group vaccinated with a needle-free intramuscular injection delivery device similar to what had been used in previous preclinical and clinical studies. All previously untested vaccination methodologies elicited improved immune responses compared to the high dose needle-free intramuscular injection delivery group. The highest neutralizing antibody responses were observed in the group that was vaccinated with the high dose formulation via intradermal electroporation. The highest IFN-γ T cell responses were also observed in the high dose intradermal electroporation group and the CD8+ T cells were the dominant contributors for the IFNγ response. Memory B cells were detected for all four serotypes. More than a year after vaccination, groups were challenged with dengue-1 virus. Both the low and high dose intradermal electroporation groups had significantly fewer days of dengue-1 virus RNAemia compared to the control group. The results from this study demonstrate that using either an electroporation device and/or the intradermal route of delivery increases the immune response generated by this vaccine in non-human primates and should be explored in humans.

Effective Protection Induced by a Monovalent DNA Vaccine against Dengue Virus (DV) Serotype 1 and a Bivalent DNA Vaccine against DV1 and DV2 in Mice.

Dengue virus (DV) is the causal pathogen of dengue fever, which is one of the most rapidly spread mosquito-borne disease worldwide and has become a severe public health problem. Currently, there is no specific treatment for dengue; thus, a vaccine would be an effective countermeasure to reduce the morbidity and mortality. Although, the chimeric Yellow fever dengue tetravalent vaccine has been approved in some countries, it is still necessary to develop safer, more effective, and less costly vaccines. In this study, a DNA vaccine candidate pVAX1-D1ME expressing the prME protein of DV1 was inoculated in BALB/c mice via intramuscular injection or electroporation, and the immunogenicity and protection were evaluated. Compared with traditional intramuscular injection, administration with 50 μg pVAX1-D1ME via electroporation with three immunizations induced persistent humoral and cellular immune responses and effectively protected mice against lethal DV1 challenge. In addition, immunization with a bivalent vaccine consisting of pVAX1-D1ME and pVAX1-D2ME via electroporation generated a balanced IgG response and neutralizing antibodies against DV1 and DV2 and could protect mice from lethal challenge with DV1 and DV2. This study sheds new light on developing a dengue tetravalent DNA vaccine.

Small-angle neutron scattering and molecular dynamics structural study of gelling DNA nanostars.

DNA oligomers with properly designed sequences self-assemble into well defined constructs. Here, we exploit this methodology to produce bulk quantities of tetravalent DNA nanostars (each one composed of 196 nucleotides) and to explore the structural signatures of their aggregation process. We report small-angle neutron scattering experiments focused on the evaluation of both the form factor and the temperature evolution of the scattered intensity at a nanostar concentration where the system forms a tetravalent equilibrium gel. We also perform molecular dynamics simulations of one isolated tetramer to evaluate the form factor numerically, without resorting to any approximate shape. The numerical form factor is found to be in very good agreement with the experimental one. Simulations predict an essentially temperature-independent form factor, offering the possibility to extract the effective structure factor and its evolution during the equilibrium gelation.

Immunization with electroporation enhances the protective effect of a DNA vaccine candidate expressing prME antigen against dengue virus serotype 2 infection

PurposeWe aimed to use the dengue virus (DV) serotype 2 as a proof of principal for testing the efficacy of a DNA vaccine candidate via in vivo electroporation in mice and rabbits prior to the development of a tetravalent vaccine. MethodsDifferent dosages of DNA pVAX1-D2ME encoding DV2 prME genes were vaccinated in mice via intramuscular injection or in vivo electroporation, immune responses and protection were determined. In DNA-vaccinated rabbits via electroporation, antibody titer and protein expression were tested. ResultsIn mice, 50μg of pVAX1-D2ME via electroporation elicited effective anti-DV2 responses and conferred significant protection against DV2 challenge. Moreover, anti-DV2 IgG and neutralizing antibodies were successfully induced in rabbits immunized with pVAX1-D2ME via electroporation and the expression of the interest protein was observed at local sites. ConclusionsEnhanced immunogenicity and protective effect conferred by pVAX1-D2ME via electroporation show great promise for the development of a dengue tetravalent DNA vaccine.

The Immunodominance Change and Protection of CD4+ T-Cell Responses Elicited by an Envelope Protein Domain III-Based Tetravalent Dengue Vaccine in Mice.

Dengue is the leading cause of mosquito-borne viral infections and no vaccine is available now. Envelope protein domain III (ED3) is the major target for the binding of dengue virus neutralizing antibodies; however, the ED3-specifc T-cell response is less well understood. To investigate the T-cell responses to four serotypes of dengue virus (DENV-1 to 4), we immunized mice using either a tetravalent ED3-based DNA or protein vaccine, or combined both as a DNA prime-protein boost strategy (prime-boost). A significant serotype-dependent IFN-γ or IL-4 response was observed in mice immunized with either the DNA or protein vaccine. The IFN-γ response was dominant to DENV-1 to 3, whereas the IL-4 response was dominant to DENV-4. Although the similar IgG titers for the four serotypes were observed in mice immunized with the tetravalent vaccines, the neutralizing antibody titers varied and followed the order of 2 = 3>1>4. Interestingly, the lower IFN-γ response to DENV-4 is attributable to the immunodominance change between two CD4+ T-cell epitopes; one T-cell epitope located at E349-363 of DENV-1 to 3 was more immunogenic than the DENV-4 epitope E313-327. Despite DENV-4 specific IFN-γ responses were suppressed by immunodominance change, either DENV-4-specific IFN-γ or neutralizing antibody responses were still recalled after DENV-4 challenge and contributed to virus clearance. Immunization with the prime-boost elicited both IFN-γ and neutralizing antibody responses and provided better protection than either DNA or protein immunization. Our findings shed light on how ED3-based tetravalent dengue vaccines sharpen host CD4 T-cell responses and contribute to protection against dengue virus.

The immunogenicity of tetravalent dengue DNA vaccine in mice pre-exposed to Japanese encephalitis or Dengue virus antigens.

Asian countries are an endemic area for both dengue (DENV) and Japanese encephalitis viruses (JEV). While JEV vaccines have been used extensively in this region, DENV vaccines remains under development. Whether preexisting naturally acquired or vaccination-induced immunity against JEV may affect the immune response to dengue vaccine candidate is unclear. In this study we used mice previously immunized with JEV vaccines to evaluate the impact on dengue-specific neutralizing antibody responses to a tetravalent dengue DNA vaccine candidate (TDNA). A tetravalent cocktail of plasmids encoding pre-membrane and envelope proteins from each dengue serotype was administered into mice which had been previously primed with inactivated or live-attenuated JEV vaccines, or dengue serotype2 virus (DENV-2). Neutralizing antibody response was measured employing a plaque reduction neutralization test at two weeks after the priming and at four weeks after the second dose of the dengue tetravalent plasmids. Inactivated or live-attenuated JEV vaccines, or DENV-2 induced low levels of neutralizing antibodies against the homologous viruses (JE and dengue virus, respectively). DENV-2 injection induced also low levels of cross-reactive antibodies against DENV-1, -3 and -4. JEV vaccines have no effect on the dengue-specific neutralizing antibody responses to the subsequent TDNA immunization. Pre-exposure to DENV-2 infection increased DENV-2 specific response neutralizing antibody to two doses of TDNA plasmids by six folds, but did not affect antibody response to other serotypes. Priming with JEV vaccines did not impact on dengue virus-specific neutralizing antibody response to a dengue TDNA vaccine candidate in mice.

Aptamer-facilitated cryoprotection of viruses.

Global vaccination and gene therapy programs have an urgent demand for stabilization of viral vectors at low temperature. We used a quadramer, a bridge-connected DNA tetra-aptamer to antivesicular stomatitis virus (VSV), as a viral cryoprotectant. Results showed that the tetravalent antivirus DNA aptamers protect viral activity during multiple freeze-thaw cycles, shield from neutralizing antibodies, and decrease aggregation of viral particles.

Induction of neutralizing antibody response against four dengue viruses in mice by intramuscular electroporation of tetravalent DNA vaccines.

DNA vaccine against dengue is an interesting strategy for a prime/boost approach. This study evaluated neutralizing antibody (NAb) induction of a dengue tetravalent DNA (TDNA) vaccine candidate administered by intramuscular-electroporation (IM-EP) and the benefit of homologous TDNA boosting in mice. Consensus humanized pre-membrane (prM) and envelope (E) of each serotypes, based on isolates from year 1962–2003, were separately cloned into a pCMVkan expression vector. ICR mice, five-six per group were immunized for three times (2-week interval) with TDNA at 100 µg (group I; 25 µg/monovalent) or 10 µg (group II; 2.5 µg/monovalent). In group I, mice received an addtional TDNA boosting 13 weeks later. Plaque reduction neutralization tests (PRNT) were performed at 4 weeks post-last immunization. Both 100 µg and 10 µg doses of TDNA induced high NAb levels against all DENV serotypes. The median PRNT50 titers were comparable among four serotypes of DENV after TDNA immunization. Median PRNT50 titers ranged 240–320 in 100 µg and 160–240 in 10 µg groups (p = ns). A time course study of the 100 µg dose of TDNA showed detectable NAb at 2 weeks after the second injection. The NAb peaked at 4 weeks after the third injection then declined over time but remained detectable up to 13 weeks. An additional homologous TDNA boosting significantly enhanced the level of NAb from the nadir for at least ten-fold (p<0.05). Of interest, we have found that the use of more recent dengue viral strain for both vaccine immunogen design and neutralization assays is critical to avoid a mismatching outcome. In summary, this TDNA vaccine candidate induced good neutralizing antibody responses in mice; and the DNA/DNA prime/boost strategy is promising and warranted further evaluation in non-human primates.

Accurate phase diagram of tetravalent DNA nanostars

We evaluate, by means of molecular dynamics simulations employing a realistic DNA coarse-grained model, the phase behaviour and the structural and dynamic properties of tetravalent DNA nanostars, i.e., nanoconstructs completely made of DNA. We find that, as the system is cooled down, tetramers undergo a gas–liquid phase separation in a region of concentrations which, if the difference in salt concentration is taken into account, is comparable with the recently measured experimental phase diagram [S. Biffi, R. Cerbino, F. Bomboi, E. M. Paraboschi, R. Asselta, F. Sciortino, and T. Bellini, Proc. Natl. Acad. Sci. U.S.A. 110, 15633 (2013)]. We also present a mean-field free energy for modelling the phase diagram based on the bonding contribution derived by Wertheim in his studies of associating liquids. Combined with mass-action law expressions appropriate for DNA binding and a numerically evaluated reference free energy, the resulting free energy qualitatively reproduces the numerical data. Finally, we report information on the nanostar structure, e.g., geometry and flexibility of the single tetramer and of the collective behaviour, providing a useful reference for future small angle scattering experiments, for all investigated temperatures and concentrations.

Gels of DNA Nanostars Never Crystallize

Using state-of-the-art numerical techniques, we show that, upon lowering the temperature, tetravalent DNA nanostars form a thermodynamically stable, fully bonded equilibrium gel. In contrast to atomic and molecular network formers, in which the disordered liquid is always metastable with respect to some crystalline phase, we find that the DNA nanostar gel has a lower free energy than the diamond crystal structure in a wide range of concentrations. This unconventional behavior, here verified for the first time in a realistic model, arises from the large arm flexibility of the DNA nanostars, a property that can be tuned by design. Our results confirm the thermodynamic stability of the recently experimentally realized DNA hydrogels.

Immunogenicity and protective efficacy of a vaxfectin-adjuvanted tetravalent dengue DNA vaccine

A prototype dengue-1 DNA vaccine was shown to be safe and immunogenic in a previous Phase 1 clinical trial. Anti-dengue-1 neutralizing antibody responses were detectable only in the group of volunteers receiving the high dose of nonadjuvanted vaccine and the antibody titers were low. Vaxfectin(®), a lipid-based adjuvant, enhances the immunogenicity of DNA vaccines. We conducted a nonhuman primate study to evaluate the effect of Vaxfectin(®) on the immunogenicity of a tetravalent dengue DNA vaccine. Animals were immunized on days 0, 28 and 84, with each immunization consisting of 3mg of Vaxfectin(®)-adjuvanted tetravalent dengue DNA vaccine. The use of Vaxfectin(®) resulted in a significant increase in anti-dengue neutralizing antibody responses against dengue-1, -3 and -4. There was little to no effect on T cell responses as measured by interferon gamma ELISPOT assay. Animals immunized with the Vaxfectin(®)-formulated tetravalent DNA vaccine showed significant protection against live dengue-2 virus challenge compared to control animals (0.75 mean days of viremia vs 3.3 days). Animals vaccinated with nonadjuvanted DNA had a mean 2.0 days of viremia. These results support further evaluation of the Vaxfectin(®)-adjuvanted tetravalent dengue DNA vaccine in a Phase 1 clinical trial.

Balance of infection-enhancing and neutralizing antibodies induced by a dengue tetravalent DNA vaccine in a mouse model

Most dengue vaccines currently under development are designed to induce neutralizing antibodies. Although neutralizing antibodies are believed to be protective, most of the antibody species that have neutralizing activities also have infection-enhancing activities at subneutralizing doses. Thus, the balance of neutralizing and enhancing activities induced by a dengue tetravalent vaccine was analyzed in this study in a DNA vaccine model using mice. The balance was measured using semi-adherent K562 cells in an assay system we previously developed. This system detected enhancing activities in sera from mice immunized with the vaccine when complement was absent from the virus–antibody mixture in this assay. Mice passively transferred with vaccinated mouse sera or monoclonal antibodies that possessed neutralizing activities as determined on Vero cells, also showed enhancing activities in their sera during the course of antibody waning in the absence of complement. However, these enhancing activities were abolished or altered to neutralizing activities in the presence of complement in our assay system, except in mice passively transferred with a monoclonal antibody of isotype IgG1. Thus, enhancing activities were not shown in mice immunized with a DNA vaccine as far as complement was included in our in vitro assay system.

Construction, purification, and evaluation of multivalent DNA vaccine against Schistosoma japonicum

DNA vaccine encoding a multivalent antigen is a novel approach of protective immunization. Four Schistosoma japonicum candidate antigen genes, glyceraldehyde-3-phosphate dehydrogenase (SjGAPDH), 23kDa transmembrane protein (Sj23), 14kDa fatty-acid binding protein (SjFABP) and 26kDa glutathione-S-transferase (Sj26), are recombined into two pieces of fusion genes SjFABP.Sj23 and Sj26.SjGAPDH, respectively. Tetravalent DNA vaccine pVIVO2-SjFABP.Sj23/Sj26.SjGAPDH is constructed by co-expressing these two fusion genes. The super-coiled DNA vaccines for large-scale clinic application were purified by sequential chromatographies including group separation chromatography and affinitive chromatographies. The purified DNA vaccines were evaluated for in vivo and in vitro transfection assay. The immunoprotective properties of the different kinds of constructed DNA vaccines were appraised by pharmacological trials. The pharmacological trials results showed that tetravalent DNA vaccine has higher protective efficiency than other tested DNA vaccines.

Effect of Pre-Existing Immunity to Flaviviruses on Balanced Induction of Neutralizing Antibodies by a Dengue Tetravalent DNA Vaccine in Mice

We previously demonstrated that a DNA-based tetravalent dengue vaccine can induce balanced neutralizing antibody responses against all four types of dengue virus (DENV1–4), using naive mice. On the other hand, individuals receiving the dengue vaccine may have previously been infected with dengue virus (DENV1–4) or vaccinated with other fl avivirus vaccines. Thus, we investigated the effect of preimmunity against fl aviviruses on immunogenicity of a dengue tetravalent DNA vaccine in mice. Mice preimmunized with DENV2 developed higher antibody levels against DENV2 than other types of dengue virus after the fi rst dengue vaccination. However, the second vaccination provided similar increases (4- to 8-fold) in antibody levels against all types of dengue virus, compared with non-preimmunized mice. Preimmunization with yellow fever or Japanese encephalitis vaccines did not affect the immunogenicity of the tetravalent vaccine, except for slight and occasional increases in antibody levels against certain types of viruses. Thus, the dengue tetravalent DNA vaccine could provide balanced induction of dengue antibody responses even in mice preimmunized with any of the three fl aviviruses, at least after the second vaccination.

Particle-mediated DNA vaccines against seasonal and pandemic influenza viruses elicit strong mucosal antibody and T cell responses in the lung

The recent outbreaks of H5N1 avian and H1N1 swine-origin influenza viruses highlight the urgent need for vaccine strategies able to confer broad host immunity against both seasonal and pandemic strains of influenza. As the lung mucosa is the first barrier against pulmonary influenza infection, an effective influenza vaccine strategy should elicit anti-viral immune responses in the respiratory tract where they may afford better protection. As a safe alternative to live attenuated vaccines, we evaluated the immunogenicity of gene gun-based immunization using a tetravalent DNA vaccine encoding the HA genes from three seasonal flu strains (H1, H3, and B) and a H5N1 pandemic strain. Vaccinated mice were found to exhibit high titers of serum antibodies specific for each of the encoded HA antigens, as well as cross-reactivity to drifted strains of virus. Importantly, significant levels of HA-specific mucosal antibodies were also detected in the lung airways of vaccinated mice. Consistent with the belief that immune responses against conserved regions of the virus may provide cross-protection against divergent strains of influenza, we also evaluated the immunogenicity of particle-mediated DNA vaccines encoding the highly conserved viral nucleoprotein (NP). Mice immunized with a DNA vaccine encoding a consensus NP sequence derived from circulating strains of virus exhibited robust NP-specific CD8 T cell responses in both spleen and lung tissue. Together, these results demonstrate the ability of skindelivered particle-mediated DNA vaccines to elicit strong systemic and mucosal immune responses against both variable and conserved influenza antigens and support the concept that particle-mediated DNA vaccines may offer an effective strategy to improve protection against seasonal and pandemic influenza.

Protection against dengue virus by non-replicating and live attenuated vaccines used together in a prime boost vaccination strategy

A new vaccination strategy for dengue virus (DENV) was evaluated in rhesus macaques by priming with tetravalent purified inactivated virus (TPIV) or tetravalent plasmid DNA vaccines expressing the structural prME gene region (TDNA) then boosting 2 months later with a tetravalent live attenuated virus (TLAV) vaccine. Both vaccine combinations elicited virus neutralizing (N) antibodies. The TPIV/TLAV combination afforded complete protection against DENV 3 challenge at month 8. In a second experiment, priming with TPIV elicited N antibodies against all four serotypes (GMT 1:28 to 1:43). Boosting with TLAV led to an increase in the GMT for each serotype (1:500 to 1:1200 for DENVs 1, 3, and 4, and greater than 1:6000 for DENV 2), which declined by month 8 (GMT 1:62 for DENV 3, 1:154 for DENV 1, 1:174 for DENV 4, and 1:767 for DENV 2). After challenge with each one of the four DENV serotypes, vaccinated animals exhibited no viremia but showed anamnestic antibody responses to the challenge viruses.

Dengue tetravalent DNA vaccine increases its immunogenicity in mice when mixed with a dengue type 2 subunit vaccine or an inactivated Japanese encephalitis vaccine

We previously developed a dengue tetravalent DNA vaccine that can induce neutralizing antibodies against four dengue viruses in mice. Here, we demonstrated that immunogenicity of our tetravalent vaccine is synergistically increased in mice by co-immunization with dengue type 2 virus (DENV2) subviral extracellular particles (D2EPs) or inactivated Japanese encephalitis vaccine (JEVAX). A single immunization with a mixture of 100 μg of the tetravalent vaccine and 150 ng of D2EPs or a 1/10 dose of JEVAX induced moderate levels of neutralizing antibodies in a 90% plaque reduction assay. Immunized mice were protected from “artificial” viremia created by intravenous injection with DENV2.