DNA Vaccine Adjuvant Research Articles

PGM-CSF as an adjuvant in DNA vaccination against SARS-CoV-2

The emergence of SARS-CoV-2 presents a significant global public health dilemma. Vaccination has long been recognized as the most effective means of preventing the spread of infectious diseases. DNA vaccines have attracted attention due to their safety profile, cost-effectiveness, and ease of production. This study aims to assess the efficacy of plasmid-encoding GM-CSF (pGM-CSF) as an adjuvant to augment the specific humoral and cellular immune response elicited by DNA vaccines based on the receptor-binding domain (RBD) antigen. Compared to the use of plasmid-encoded RBD (pRBD) alone, mice that were immunized with a combination of pRBD and pGM-CSF exhibited significantly elevated levels of RBD-specific antibody titers in serum, BALF, and nasal wash. Furthermore, these mice generated more potent neutralization antibodies against both the wild-type and Omicron pseudovirus, as well as the ancestral virus. In addition, pGM-CSF enhanced pRBD-induced CD4+ and CD8+ T cell responses and promoted central memory T cells storage in the spleen. At the same time, tissue-resident memory T (Trm) cells in the lung also increased significantly, and higher levels of specific responses were maintained 60 days post the final immunization. pGM-CSF may play an adjuvant role by promoting antigen expression, immune cells recruitment and GC B cell responses. In conclusion, pGM-CSF may be an effective adjuvant candidate for the DNA vaccines against SARS-CoV-2.

Immune-enhancing effects of Astragalus polysaccharides and Ganoderma lucidum polysaccharides on Vibrio harveyi flgJ DNA vaccine in grouper.

Astragalus polysaccharides (APS) and Ganoderma lucidum polysaccharides (GLP) have been shown to possess strong immunoregulatory properties in aquatic animals. In this study, the fragment containing Vibrio harveyi flgJ gene was ligated into pcDNA3.1(+) vector and pcDNA3.1(+)-flgJ was constructed as DNA vaccine. APS and GLP were used as DNA vaccine adjuvants to evaluate the immunoregulatory effect by intramuscular injection to pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂E. lanceolatus). The results showed that pcDNA3.1(+)-flgJ combined with APS or GLP could significantly up-regulate the innate and adaptive immune response in fish, including serum-specific antibody titres, catalase and lysozyme activities. At the same time, DNA vaccine combined with APS or GLP significantly up-regulated the expression levels of CD8α, IgM, IL-1β, MHC-Iα, MyD88 and TLR3 genes in thymus, head kidney, spleen and liver of pearl gentian grouper in comparison with those of the pFlgJ group. After 42 days post-vaccination, V. harveyi was used to challenge pearl gentian grouper by intraperitoneal injection. The relative percentage of survival (RPS) of pFlgJ, pFlgJ +APS, pFlgJ +GLP and pFlgJ+APS+GLP groups were 69%, 81%, 77% and 88%, respectively. These results suggested APS and GLP were potential adjuvants for DNA vaccine against V. harveyi infection in pearl gentian grouper.

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-1β). 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-1β maturation and IL-1β 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.

Foot and Mouth Disease Vaccine Development and Challenges in Inducing Long-Lasting Immunity: Trends and Current Perspectives.

Foot and mouth disease (FMD) is an extremely contagious viral disease of livestock caused by foot and mouse disease virus genus: Aphthovirus, which causes a serious economic impact on both individual farmers and the national economy. Many attempts to advance a vaccine for FMD have failed to induce sterile immunity. The classical methods of vaccine production were due to selective accumulation of mutations around antigenic and binding sites. Reversion of the agent by positive selection and quasi-species swarm, use of this method is inapplicable for use in non-endemic areas. Chemical attenuation using binary ethyleneimine (BEI) protected the capsid integrity and produced a pronounced immunity against the challenge strain. Viral antigens which have been chemically synthesized or expressed in viruses, plasmid, or plants were tried in the vaccination of animals. DNA vaccines expressing either structural or nonstructural protein antigens have been tried to immunize animals. Using interleukins as a genetic adjuvant for DNA vaccines have a promising effect. While the challenges of inducing sterile immunity lies on non-structural (NS) proteins of FMDV which are responsible for apoptosis of dendritic cells and have negative effects on lympho-proliferative responses which lead to transient immunosuppression. Furthermore, destruction of host protein trafficking by nonstructural proteins suppressed CD8+ T-cell proliferation. In this review, it tried to address multiple approaches for vaccine development trials and bottle necks of producing sterile immunity.

Intranasal immunization with O-2\u2032-Hydroxypropyl trimethyl ammonium chloride chitosan nanoparticles loaded with Newcastle disease virus DNA vaccine enhances mucosal immune response in chickens

BackgroundThere has been a great interest in developing strategies for enhancing antigen delivery to the mucosal immune system as well as identifying mucosal active immunostimulating agents. To elevate the potential of O-2ʹ-Hydroxypropyl trimethyl ammonium chloride chitosan (O-2ʹ-HACC) as an adjuvant and mucosal immune delivery carrier for DNA vaccine, we prepared the O-2ʹ-HACC loaded with Newcastle disease virus (NDV) F gene plasmid DNA and C3d6 molecular adjuvant (O-2ʹ-HACC/pFDNA microparticles).ResultsThe O-2ʹ-HACC/pFDNA exhibited a regular spherical morphology with a particle size of 202.3 ± 0.52 nm, a zeta potential of 50.8 ± 8.21 mV, encapsulation efficiency of 90.74 ± 1.10%, and a loading capacity of 49.84 ± 1.20%. The plasmid DNA could be sustainably released from the O-2ʹ-HACC/pFDNA after an initial burst release. Intranasal vaccination of chickens immunized with O-2ʹ-HACC/pFDNA not only induced higher anti-NDV IgG and sIgA antibody titers but also significantly promoted lymphocyte proliferation and produced higher levels of IL-2, IL-4, IFN-γ, CD4+, and CD8 + T lymphocytes compared with the NDV commercial live attenuated vaccine. Intranasal delivery of the O-2ʹ-HACC/pFDNA enhanced humoral, cellular, and mucosal immune responses and protected chickens from the infection of highly virulent NDV compared with the intramuscular delivery.ConclusionsCollectively, our findings indicated that the O-2ʹ-HACC could be used as a vaccine adjuvant and delivery system for mucosal immunity and have an immense application promise.Graphic

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.

The Function of the PRRSV-Host Interactions and Their Effects on Viral Replication and Propagation in Antiviral Strategies.

Porcine reproductive and respiratory syndrome virus (PRRSV) affects the global swine industry and causes disastrous economic losses each year. The genome of PRRSV is an enveloped single-stranded positive-sense RNA of approximately 15 kb. The PRRSV replicates primarily in alveolar macrophages of pig lungs and lymphatic organs and causes reproductive problems in sows and respiratory symptoms in piglets. To date, studies on how PRRSV survives in the host, the host immune response against viral infections, and pathogenesis, have been reported. PRRSV vaccines have been developed, including inactive virus, modified live virus, attenuated live vaccine, DNA vaccine, and immune adjuvant vaccines. However, there are certain problems with the durability and effectiveness of the licensed vaccines. Moreover, the high variability and fast-evolving populations of this RNA virus challenge the design of PRRSV vaccines, and thus effective vaccines against PRRSV have not been developed successfully. As is well known, viruses interact with the host to escape the host’s immune response and then replicate and propagate in the host, which is the key to virus survival. Here, we review the complex network and the mechanism of PRRSV–host interactions in the processes of virus infection. It is critical to develop novel antiviral strategies against PRRSV by studying these host–virus interactions and structures to better understand the molecular mechanisms of PRRSV immune escape.

ATP stabilised and sensitised calcium phosphate nanoparticles as effective adjuvants for a DNA vaccine against cancer.

Cancer vaccines based on DNA encoding oncogenes have shown great potential in preclinical studies. However, the efficacy of DNA vaccines is limited by their weak immunogenicity because of low cellular internalisation and insufficient activation of dendritic cells (DCs). Calcium phosphate (CP) nanoparticles (NPs) are biodegradable vehicles with low toxicity and high loading capacity of DNA but suffer from stability issues. Here we employed adenosine triphosphate (ATP) as a dual functional agent, i.e. stabiliser for CP and immunological adjuvant, and applied the ATP-modified CP (ACP) NPs to the DNA vaccine. ACP NP-enhanced cellular uptake and improved transfection efficiency of DNA vaccine, and further showed the ability to activate DCs that are critical for them to prime T cells in cancer immunotherapy. As a result, a higher level of antigen-specific antibody with stronger tumour growth inhibition was achieved in mice immunised with the ACP-DNA vaccine. Overall, this one-step synthesised ACP NPs are an efficient nano-delivery system and nano-adjuvant for cancer DNA vaccines.

The Combination of Molecular Adjuvant CCL35.2 and DNA Vaccine Significantly Enhances the Immune Protection of Carassius auratus gibelio against CyHV-2 Infection.

Cyprinid herpesvirus 2 (CyHV-2) infection results in huge economic losses in gibel carp (Carassius auratus gibelio) industry. In this study, we first constructed recombinant plasmids pcORF25 and pcCCL35.2 as DNA vaccine and molecular adjuvant against CyHV-2, respectively, and confirmed that both recombinant plasmids could be effectively expressed in vitro and in vivo. Then, the vaccination and infection experiments (n = 50) were set as seven groups. The survival rate (70%) in ORF25/CCL35.2 group was highest. The highest specific antibody levels were found in ORF25/CCL35.2 group in major immune tissues by qRT-PCR, and confirmed in serum by ELISA assay, antibody neutralization titer, and serum incubation-infection experiments. Three crucial innate immune indices, namely C3 content, lysozyme, and total superoxide dismutase (TSOD) activities, were highest in ORF25/CCL35.2 group in serum. pcORF25/pcCCL35.2 can effectively up-regulate mRNA expressions of some important immune genes (IL-1β, IL-2, IFN-γ2, and viperin), and significantly suppress CyHV-2 replication in head kidney and spleen tissues. The minimal tissue lesions can be seen in ORF25/CCL35.2 group in gill, spleen, and trunk kidney tissues by histopathological examination. The results indicated that the combination of DNA vaccine pcORF25 and molecular adjuvant pcCCL35.2 is an effective method against CyHV-2 infection, suggesting a feasible strategy for the control of fish viral diseases.

Molecular Adjuvants for DNA Vaccines: Application, Design, Preparation, and Formulation.

Compared with conventional vaccines, the main advantage of DNA vaccine-based methods is its continued expression of the plasmid-encoded antigens followed by the induction of subsequent humoral and cellular immunities. DNA vaccines are currently used in animal models, but limited success has been obtained for use in clinical applications due to their poor immunogenicity. Various strategies are attempted to improve the induced immune response of DNA vaccines. It has been demonstrated that co-administration of molecular adjuvants with DNA vaccines is a promising approach to effectively elicit protective immunity by increasing the transfection efficiency of DNA vaccines. Genetic adjuvants are incorporated to promote activation of the transfected local antigen-presenting cells (APCs) and immune cells in the draining lymph node and polarization of T-cell subsets to decrease T-cell tolerance to the specific antigen. Here we provide an overview of different types of genetic adjuvants. The aim of the current chapter is to present a framework for the construction of a gene-based vaccine and adjuvant. Moreover, we describe the application of DNA vaccines co-administered with different types of genetic adjuvants and the methods to evaluate their potency in the mouse models.

The Promotion of Peony Seed Proteolysis Product to the Immune Efficacy Induced by ptfA Gene DNA Vaccine of Avian Pasteurella multocida

Background: Avian Pasteurella multocida is one of the pathogens that affect the health of poultry. The protective efficacy of traditional attenuated vaccine is not ideal. In previous study, we prepared ptfA gene DNA vaccine of avian P. multocida. However, the protective effect of ptfA gene DNA vaccine was inferior to the attenuated vaccine. Therefore, it is necessary to improve the immune efficacy of avian P. multocida DNA vaccine, such as screening for novel adjuvant. Methods: In this study, the peony seed proteolysis product was gavaged to chickens before DNA vaccination or was added to the ptfA gene DNA vaccine as adjuvant. These vaccines were administered to chickens and the serum antibody, lymphocyte proliferation levels, IFN-g, IL-2, IL-4 and IL-6 concentrations secreted by peripheral blood lymphocytes were determined. After challenging with virulent avian P. multocida, survival time and protective efficacy was evaluated. Result: Following vaccination, no significant differences in antibody levels and concentrations of IL-4 among the DNA vaccine group, adjuvant-DNA vaccine group and gavaged group were observed. The stimulation index (SI) values, concentrations of IFN-g, IL-2 and IL-6 in adjuvant-DNA vaccine group and gavaged group were significantly higher than those in the DNA vaccine group. The protective efficacy of live attenuated vaccine group, DNA vaccine group, adjuvant-DNA vaccine group and gavaged group were 92%, 52%, 72% and 60%, respectively. This study has laid a foundation for the design and application of future DNA vaccines of P. multocida and DNA vaccine adjuvant.

An IL-6 gene in humphead snapper (Lutjanus sanguineus): Identification, expression analysis and its adjuvant effects on Vibrio harveyi OmpW DNA vaccine

Interleukin 6 (IL-6) is a pleiotropic cytokine that plays important role in mediating the innate and adaptive immune responses against pathogen infection. In this study, an IL-6 homolog (Ls-IL6) was identified and characterized from humphead snapper, Lutjanus sanguineus. The full-length cDNA of Ls-IL6 was 1066 bp, containing an open reading frame (ORF) of 639 bp encoding 212 amino acids, 5′ untranslated region(UTR) of 63 bp and 3’ UTR of 605 bp. The predicted Ls-IL6 protein had typical motif of IL-6 family and shared high identities to teleost IL-6s. Ls-IL6 extensively expressed in various tissues, and the highest expression of Ls-IL6 was detected in head kidney, spleen and thymus. In vivo, the transcript levels of Ls-IL6 were significantly up-regulated in response to Vibrio harveyi infection. Moreover, the DNA plasmid containing the OmpW of V. harveyi together with the gene encoding Ls-IL6 were successfully constructed and administered to fish, the protective efficacy of Ls-IL6 was investigated. Compared with the pcDNA-OmpW group, the level of specific antibodies against V. harveyi increased in pcDNA-IL6-OmpW injected group. After V. harveyi infection, the pcDNA-IL6-OmpW vaccinated fish showed higher relative percent survival (76%) than the relative survival of fish immunized with pcDNA-OmpW (60%). These results indicated that Ls-IL6 was involved in immune response against V. harveyi infection and could be applied as a promising adjuvant for DNA vaccines against V. harveyi.

Simultaneous delivery of DNA vaccine and hydrophobic adjuvant using reducible polyethylenimine-functionalized graphene oxide for activation of dendritic cells

The activation and maturation of dendritic cells are critical in immunotherapy, and the potency of DCs is associated with high levels of antigen presentation on major histocompatibility complex (MHC) class I and II and the expression of costimulatory signals. Graphene oxide (GO) and its derivatives have many excellent physicochemical properties, including large and hydrophobic surface available for interacting with hydrophobic or aromatic drugs via π–π stacking forces, flexibility of the chemical modification on the surfaces, and capacity of DC activation. In this study, we designed a simple strategy to achieve the co-delivery of a DNA vaccine and hydrophobic immune adjuvant (R848) and to enhance the adjuvanticity of R848 via the synergistic effect of GO and R848. Thiolated low-molecular-weight polyethylenimine (TPEI1.8) was crosslinked with 4-aminothiophenol-modified GO (TGO) via the formation of disulfide bonds. Thus, TGO with its assembled TPEI1.8 could not only load R848 but also electrostatically interact with the DNA vaccine. Owing to the reducibility of the disulfide bond in the cellular environment, the DNA vaccine could be readily released. This system can significantly enhance the DNA transfection, the expression of the costimulatory signal, and the level of antigen presentation to MHC class I DCs for their activation and maturation.

Generation and functional evaluation of a DNA vaccine co-expressing Vibrio anguillarum VAA protein and flounder interleukin-2

In our previous study, a DNA plasmid encoding the VAA gene of Vibrio anguillarum was constructed and demonstrated to confer moderated protection against V. anguillarum challenge. Here, a bicistronic DNA vaccine (pVAA-IRES-IL2), co-expressing the VAA gene of V. anguillarum and Interleukin-2 (IL2) gene of flounder, was constructed to increase the protective efficacy of VAA DNA vaccine. The potential of pVAA-IRES-IL2 to express both VAA and IL2 in transfected HINAE cell lines was confirmed by immunofluorescence assay. Further, the variation of sIgM+, CD4-1+, CD4-2+ lymphocytes and production of VAA-specific antibodies in flounder, which was intramuscularly immunized with three DNA plasmids (pIRES, pVAA-IRES, pVAA-IRES-IL2), were investigated, respectively. The bacterial burden and relative percentage survival (RPS) of flounder exposed to V. anguillarum infection were both analyzed to evaluate the efficacy of bicistronic DNA plasmid. Our results revealed that the percentages of sIgM+, CD4-1+, CD4-2+ lymphocytes and antibodies specific to VAA were remarkably increased in pVAA-IRES or pVAA-IRES-IL2 immunized fish. Moreover, the co-expression of IL2 enhanced the immune response in response to VAA DNA vaccination, as shown by the higher percentages of sIgM+, CD4-1+, CD4-2+ lymphocytes and production of specific antibody. Importantly, the RPS in pVAA-IRES-IL2 and pVAA-IRES groups reached 64.1% and 51.3%, respectively, when compared with the 97.5% cumulative mortality in pIRES group. Furthermore, the number of V. anguillarum in liver, spleen and kidney of pVAA-IRES or pVAA-IRES-IL2 immunized flounder after V. anguillarum challenge was significantly reduced, as compared to that in pIRES group. These suggest that the bicistronic DNA vaccine can be an effective immunization strategy in inducing immune response against V. anguillarum infection and IL2 has the potential as the adjuvant for VAA DNA vaccine.

Effect of miR-155 as a molecular adjuvant of DNA vaccine against VHSV in olive flounder (Paralichthys olivaceus)

Rhabdoviral G protein-based DNA vaccines have been recognized as a useful way to protect cultured fish from rhabdoviral diseases. In Korea, viral hemorrhagic septicemia virus (VHSV) genotype IVa has been the primary culprit of high mortalities of cultured olive flounder (Paralichthys olivaceus). In this study, we inserted a miR-155-expressing cassette into the VHSV's G protein-based DNA vaccine, and analyzed the effects of miR-155 on the antiviral activity and on the vaccine efficacy in olive flounder. Olive flounder fingerlings were intramuscularly (i.m.) immunized with 10 μg/fish (1st experiment) or 1 μg/fish (2nd experiment) of DNA vaccine plasmids. However, there were no significant differences in mortalities and serum neutralization titers between fish immunized with 1 μg and 10 μg plasmids/fish, suggesting that i.m. injection with 1 μg plasmids/fish would be enough to induce effective adaptive immune responses in olive flounder fingerlings. In survival rates, as fish immunized with just G protein expressing plasmids showed no or too low mortalities, the adjuvant effect of miR-155 was not discernible. Also, in the serum neutralization activities, although G gene or G gene plus miR-155 expressing DNA vaccines induced significantly higher activities than control vaccines (PBS and vacant vector), no significant differences were found between G gene alone and G gene plus miR-155 expressing DNA vaccines. In the serum virucidal activity, fish immunized with G gene plus miR-155 expressing DNA vaccine showed significantly higher activity against hirame rhabdovirus (HIRRV) at 3 days post-immunization (d.p.i.) compared to other groups, suggesting that miR-155 produced from the vector can enhance innate immune responses in olive flounder. The significantly enhanced serum virucidal activities against VHSV especially at 28 d.p.i. in the groups immunized with G gene alone and G gene plus miR-155 expressing DNA vaccines reflect the increased antibodies against G protein, which could activate the classical complement pathway and subsequent viral inactivation. As the available information on the DNA vaccines in olive flounder is not sufficient, more diverse researches on the protective efficacy of DNA vaccines are needed to make more practical use of DNA vaccines in olive flounder farms.

The adjuvant effect of C60(OH)22 nanoparticles promoting both humoral and cellular immune responses to HCV recombinant proteins

Hepatitis c virus (HCV) infection is one of major causes for chronic liver diseases worldwide and could lead to death. Development of effective HCV vaccines is a powerful auxiliary method of existing treatments. Adjuvants are necessary for modern vaccines to promote immune responses. Among the various nanomaterials that have been developed, multihydroxylated fullerene (C60(OH)22) has been proved as an efficient adjuvant for human immunodeficiency virus DNA vaccine. Here, we utilized three types of HCV recombinant proteins as antigens to investigate the activity of C60(OH)22 as a protein vaccine adjuvant. The proteins were carried by C60(OH)22 in a way of surface adsorption and self-assemble encapsulation. C60(OH)22 at a relatively low dose was sufficient to promote both humoral and cellular immune responses to HCV protein antigens and reduce the usage of antigen. These results demonstrated the positive adjuvant properties of C60(OH)22 when applied to protein vaccines.

DNA prime-protein boost vaccine encoding HLA-A2, HLA-A24 and HLA-DR1 restricted epitopes of CaNA2 against visceral leishmaniasis.

Visceral leishmaniasis is a tropical and neglected disease with an estimated 200000-400000 cases and 60000 deaths worldwide each year. Currently, no clinically valid vaccine is available for this disease. In this study, we formulated DNA and protein vaccines encoding HLA-A2, HLA-A24 and HLA-DR1 restricted epitopes of CaNA2 against visceral leishmaniasis. We predicted the secondary and tertiary structures, surface properties, subcellular localization, potential binding sites and HLA-A2, HLA-A24 and HLA-DR1 restricted epitopes of CaNA2. The best candidate CpG ODN (2395, M362, D-SL03 or 685) was screened out as a DNA vaccine adjuvant. We also prepared Kmp-11 and Kmp-11/CaNA2 DNA and protein vaccines, respectively, for comparison. BALB/c mice were immunized with a DNA prime-protein boost immunization strategy and challenged with a newly isolated Leishmania strain from an individual with visceral leishmaniasis. The IgG antibody titers showed that our vaccine had strong immunogenicity with a long duration, especially cellular immunity. The spleen parasite burden of each group demonstrated that the CaNA2 vaccine had a certain immune protective effect on visceral leishmaniasis in BALB/c mice, and the amastigote reduction rate reached 76%. Preliminary safety tests confirmed the safety of the vaccine. Our work demonstrates that the HLA-A2, HLA-A24 and HLA-DR1 restricted epitope CaNA2 DNA prime-protein boost vaccine may be a safe and effective epitope vaccine candidate against visceral leishmaniasis.

Immunomodulatory activity of 2-deoxyguanosine nanoparticles in DNA vaccines

Abstract DNA vaccines induce protective immune responses against several pathogens and tumor cells, but strategies to improve their effectiveness are still under study. Only two adjuvants have been licensed by the FDA, which increases the needs for the discovery of novel adjuvants for HIV vaccines and other infectious diseases. In this regard, we study the immunomodulatory properties of 2-Deoxyguanosine-based nanoparticles (SHS) in DNA vaccines coding for HIV Gag (NL4-3 clone) and Vaccinia Virus Western Reserve (VVWR) A27 proteins. The sequence of A27L was codon optimized and linked to the Outer Domain-1 (OD1) of gp120 HIV-1 protein sequence serving as an additional molecular adjuvant. In this study, female Balb/c mice were immunized three times intramuscularly with plasmids (pGag and pOD1-A27L). Then, seven days after the last dose, cellular and humoral immunity were evaluated through ELISpot and ELISA assays. We found significantly higher frequencies of IFN-γ-producing cells in mouse immunized with pGag and pOD1-A27L co-formulated with SHS versus controls. We also found higher significant levels of IFN-γ over IL-4 in supernatant of splenocytes isolated from mice receiving pGag and pOD1-A27L combined with SHS. Moreover, administration of pOD1-A27L+SHS stimulated a significant higher production of total IgG in mice when compared to Naïve group. Here, we demonstrated that pGag and pOD1-A27L vaccines co-formulated with SHS nanoparticles successfully activate adaptive immunity through production of Th-1 cytokines and/or IgG antibodies. As this type of immunity is essential to clear many viral infections, these data support the evaluation of SHS nanoparticles as a novel adjuvant for DNA vaccines against multiple viral infectious agents.

Transcriptome analysis of plasmid-induced genes sheds light on the role of type I IFN as adjuvant in DNA vaccine against infectious salmon anemia virus.

A previous study showed that a plasmid expressing IFNa (pIFNa) strongly enhanced protection and antibody production of a DNA vaccine against infectious salmon anemia virus (ISAV) in Atlantic salmon. The vaccine consisted of a plasmid (pHE) expressing the virus hemagglutinin-esterase as an antigen. To increase the understanding of the adjuvant effect of pIFNa, we here compared transcriptome responses in salmon muscle at the injection site at week 1 and 2 after injection of pIFNa, pHE, plasmid control (pcDNA3.3) and PBS, respectively. The results showed that the IFNa plasmid mediates an increase in gene transcripts of at least three major types in the muscle; typical IFN-I induced genes (ISGs), certain chemokines and markers of B- cells, T-cells and antigen-presenting cells. The latter suggests recruitment of cells to the plasmid injection site. Attraction of lymphocytes was likely caused by the induction of chemokines homologous to mammalian CCL5, CCL8, CCL19 and CXCL10. IFN may possibly also co-stimulate activation of lymphocytes as suggested by studies in mammals. A major finding was that both pcDNA3.3 and pHE caused responses similar to pIFNa, but at lower magnitude. Plasmid DNA may thus by itself have adjuvant activity as observed in mammalian models. Notably, pHE had a lower effect on many immune genes including ISGs and chemokines than pcDNA3.3, which suggests an inhibitory effect of HE expression on the immune genes. This hypothesis was supported by an Mx-reporter assay. The present study thus suggests that a main role for pIFNa as adjuvant in the DNA vaccine against ISAV may be to overcome the inhibitory effect of HE- expression on plasmid-induced ISGs and chemokines.

Chicken IL-7 as a potent adjuvant enhances IBDV VP2 DNA vaccine immunogenicity and protective efficacy

Our previous work has demonstrated that the mammalian interleukin-7 (IL-7) gene can enhance the immunogenicity of DNA vaccine. Whether chicken IL-7 (chIL-7) possesses the ability to enhance the immunogenicity of VP2 DNA vaccine of infectious bursal disease virus (IBDV) remained unknown. To investigate this, we constructed a VP2 antigenic region (VP2366) gene and chIL-7 gene vectors, co-immunized chicken with these vectors and analyzed the effects of the chIL-7 gene on VP2366 gene immunogenicity. Results showed that co-administrated chIL-7 gene with VP2 DNA vaccine significantly increased specific serum antibody titers against IBDV, and enhanced lymphocyte proliferation and IFN-γ and IL-4 productions. More importantly, chIL-7 gene significantly increased VP2366 gene-induced protection against virulent IBDV infection, indicating that the chIL-7 gene possessed the capacity to enhance VP2366 DNA vaccine immunogenicity, and therefore might function as a novel adjuvant for IBDV VP2 DNA vaccine. Mechanically, chIL-7 could stimulate the common cytokine receptor γ chain (γc) expressions in vitro and in vivo, which might be involved in chIL-7 enhancement of the immunogenicity of VP2 DNA vaccine.