DNA Vaccine Adjuvant Research Articles

Co-delivery of ccl19 gene enhances anti-caries DNA vaccine pCIA-P immunogenicity in mice by increasing dendritic cell migration to secondary lymphoid tissues

To investigate how co-delivery of the gene encoding C-C chemokine ligand-19 (CCL-19) affected the systemic immune responses to an anti-caries DNA vaccine pCIA-P in mice. Plasmid encoding CCL19-GFP fusion protein (pCCL19/GFP) was constructed by inserting murine ccl19 gene into GFP-expressing vector pAcGFP1-N1. Chemotactic effect of the fusion protein on murine dendritic cells (DCs) was assessed in vitro and in vivo using transwell and flow cytometric analysis, respectively. BALB/c mice were administered anti-caries DNA vaccine pCIA-P plus pCCL19/GFP (each 100 μg, im) or pCIA-P alone. Serum level of anti-PAc IgG was assessed with ELISA. Splenocytes from the mice were stimulated with PAc protein for 48 h, and IFN-γ and IL-4 production was measured with ELISA. The presence of pCCL19/GFP in spleen and draining lymph nodes was assessed using PCR. The expression of pCCL19/GFP protein in these tissues was analyzed under microscope and with flow cytometry. The expression level of CCL19-GFP fusion protein was considerably increased 48 h after transfection of COS-7 cells with pCCL19/GFP plasmids. The fusion protein showed potent chemotactic activity on DCs in vitro. The level of serum PAc-specific IgG was significantly increased from 4 to 14 weeks in the mice vaccinated with pCIA-P plus pCCL19/GFP. Compared to mice vaccinated with pCIA-P alone, the splenocytes from mice vaccinated with pCIA-P plus pCCL19/GFP produced significantly higher level of IFN-γ, but IL-4 production had no significant change. Following intromuscular co-delivery, pCCL19/GFP plasmid and fusion protein were detected in the spleen and draining lymph nodes. Administration of CCL19 gene in mice markedly increased the number of mature DCs in secondary lymphoid tissues. CCL19 serves as an effective adjuvant for anti-caries DNA vaccine by inducing chemotactic migration of DCs to secondary lymphoid tissues.

Clitocybe nuda Activates Dendritic Cells and Acts as a DNA Vaccine Adjuvant.

This work represents the first evaluation of the effects of water extract of C. nuda (WE-CN), an edible mushroom, on murine bone marrow-derived dendritic cells (BMDCs) and the potential pathway through which the effects are mediated. Our experimental results show that WE-CN could induce phenotypic maturation of DCs, as shown by the increased expression of MHC and costimulatory molecules. In addition, it also induced the proinflammatory cytokines expression on DCs and enhanced both the proliferation and IFN-γ secretion of allogenic T cells. Therefore, since WE-CN did not induce maturation of DCs generated from mice with mutated TLR-4 or TLR-2, suggesting that TLR4 and TLR2 might function as membrane receptors for WE-CN. Moreover, the mechanism of action of WE-CN may be mediated by increased phosphorylation of ERK, p38, and JNK mitogen-activated protein kinase (MAPK) and increased NF-κB p65 activity, which are important signaling molecules downstream of TLR-4 and TLR-2. Finally, coimmunization of mice with WE-CN and a HER-2/neu DNA vaccine induced a HER-2/neu-specific Th1 response that resulted in significant inhibition of HER-2/neu overexpressing mouse bladder tumor (MBT-2) growth. These data suggest that WE-CN induces DC maturation through TLR-4 and/or TLR-2 and that WE-CN can be used as an adjuvant in cancer vaccine immunotherapy.

MDA5 Can Be Exploited as Efficacious Genetic Adjuvant for DNA Vaccination against Lethal H5N1 Influenza Virus Infection in Chickens

Chickens lack the retinoic acid-inducible gene I (RIG-I) and sense avian influenza virus (AIV) infections by means of the melanoma differentiation-associated gene 5 product (chMDA5). Plasmid-driven expression of the N-terminal half of chMDA5 containing the caspase activation and recruitment domains [chMDA5(1-483)] triggers interferon-β responses in chicken cells. We hypothesized that mimicking virus infection by chMDA5(1-483) expression may enhance vaccine-induced adaptive immunity. In order to test this, the potential genetic adjuvant properties of chMDA5(1-483) were evaluated in vivo in combination with a suboptimal quantity of a plasmid DNA vaccine expressing haemagglutinin (HA) of H5N1 AIV. Co-administration of the HA plasmid with plasmid DNA for chMDA5(1-483) expression resulted in approximately 10-fold higher HA-specific antibody responses than injection of the HA plasmid mixed with empty vector DNA as control. Accordingly, compared with HA DNA vaccination alone, the chMDA5(1-483)-adjuvanted HA DNA vaccine mediated enhanced protection against a lethal H5N1 challenge infection in chickens, with reduced clinical signs and cloacal virus shedding. These data demonstrate that innate immune activation by expression of signaling domains of RIG-I-like receptors can be exploited to enhance vaccine efficacy.

An endogenous immune adjuvant released by necrotic cells for enhancement of DNA vaccine potency.

Improving vaccine potency in the induction of a strong cell-mediated cytotoxicity can enhance the efficacy of vaccines. Necrotic cells and the supernatant of necrotic tumor cells are attractive adjuvants, on account of their ability to recruit antigen-presenting cells to the site of antigen synthesis as well as its ability to stimulate the maturation of dendritic cells. To evaluate the utility of supernatant of necrotic tumor cells as a DNA vaccine adjuvant in a murine model. The supernatant of EL4 necrotic cells was co-administered with a DNA vaccine expressing the glycoprotein B of Herpes simplex virus-1 as an antigen model under the control of Cytomegalovirus promoter. C57BL/6 mice were vaccinated three times at two weeks intervals with glycoprotein B DNA vaccine and supernatant of necrotic EL4 cells. Five days after the last immunization, cell cytotoxicity, IFN-γ and IL-4 were evaluated. The obtained data showed that the production of IFN-γ from the splenocytes after antigenic stimulation in the presence of the supernatant of necrotic EL4 cells was significantly higher than the other groups (p<0.002). The flow cytometry results showed a significant increase in the apoptosis/necrosis of EL4 cells in the mice immunized with DNA vaccine and supernatant of necrotic EL4 cells comparing to the other groups (p<0.001). The supernatant of necrotic cells contains adjuvant properties that can be considered as a candidate for tumor vaccination.

Enhancement of HIV-1 DNA vaccine immunogenicity by BCG-PSN, a novel adjuvant

Although the importance of DNA vaccines, especially as a priming immunization has been well established in numerous HIV vaccine studies, the immunogenictiy of DNA vaccines is generally moderate. Novel adjuvant is in urgent need for improving the immunogenicity of DNA vaccine. Polysaccharide and nucleic acid fraction extracted by hot phenol method from Mycobacterium bovis bacillus Calmette-Guérin, known as BCG-PSN, is a widely used immunomodulatory product in China clinical practice. In this study, we evaluated whether the BCG-PSN could serve as a novel adjuvant of DNA vaccine to trigger better cellular and humoral immune responses against the HIV-1 Env antigen in Balb/C mouse model. The BCG-PSN was mixed with 10μg or 100μg of pDRVI1.0gp145 (HIV-1 CN54 gp145 gene) DNA vaccine and intramuscularly immunized two or three times. We found that BCG-PSN could significantly improve the immunogenicity of DNA vaccine when co-administered with DNA vaccine. Further, at the same vaccination schedule, BCG-PSN co-immunization with 10μg DNA vaccine could elicit cellular and humoral immune responses which were comparable to that induced by 100μg DNA vaccine alone. Moreover, our results demonstrate that BCG-PSN can activate TLR signaling pathways and induce Th1-type cytokines secretion. These findings suggest that BCG-PSN can serve as a novel and effective adjuvant for DNA vaccination.

Evaluation of the enhancing ability of three adjuvants for DNA vaccination using the porcine circovirus type 2 ORF2 (capsid) gene in mice

Molecular adjuvants were used to augment the amplitude of the immune response in many studies recently. Ubiquitin (ub), the peptide binding truncated C-terminal portion of heat shock protein 70 (hsp70c) and interleukin-2 (IL-2) are widely investigated adjuvants which have been proved to be efficient. In our study, we compared the enhancing ability of these three adjuvants based on DNA vaccination using the porcine circovirus type 2 ORF2 (capsid) gene in mice. The results of lymphocyte proliferation assay, flow cytometric analysis (FCM), antibody titer and cytokine production showed that ub conjugated plasmid induced a stronger Th1 type cellular immune response and an observably higher level of Cap-specific serum immunoglobulin G antibody compared with hsp70c or IL-2 conjugated plasmids during the period of post-immunization. Meanwhile, the ub conjugation vaccinated group elicited stronger specific immunity against PCV2 challenge than the others during most of the time of post-challenge. Thus, these data indicate that ub is a superior adjuvant for a PCV2 DNA vaccination than the hsp70c and IL-2 molecules.

Fusion of CTA and CTB gene to immunogen significantly enhances the immunogenicity of DNA vaccine

Background Cholera toxin and its two subunits (CTA,CTB) have been intensively investigated as mucosal adjuvants for protein based vaccine. In this study we evaluated the adjuvanticity of CTA and CTB in modality of either mixing their encoding plasmids with DNA vaccine or fusing their encoding genes to immunogen encoding gene. Methods DNA and recombinant vaccinia vaccines expressing HIV-1 AE strain tat, rev, intergrase(C-half), vif, nef fusion gene (designated as TRIVN) have been constructed. For the construction of fusion gene of CTA/CTB to TRIVN, overlapping PCR was employed to link CTA/CTB and TRIVN gene, the fused genes(TRIVN-CTA and TRIVN-CTB)were cloned into eukaryotic expression plasmid vector(pSV1.0). Six groups of female BALB/c mice were immunized with mock control, pSV-TRIVN, pSV-TRIVN-CTA, pSVTRIVN-CTB, pSV-TRIVN mixed with CTA or with CTB respectively in a DNA priming-recombinant vaccinia boosting regimen. Two weeks after the final injection, mice splenocytes were collected and IFN-g ELISPOT assay were used as readout for specific T cell response. Statistical analysis was performed by using Prism5.0 software. Results Our data showed that all constructed plasmids are capable of efficiently expressing their inserted genes. All groups immunized with vaccines raised significant more T-cell response than mock control. T-cell responses elicited by pSV-TRIVN-CTB(1548±330SFCs/106splenocytes) and pSV-TRIVN-CTA (1642±514SFCs/106splenocytes) were significantly higher than that by pSV1.0-TRIVN (520±150SFCs/106splenocytes), pSV1.0-TRIVN mixed with CTA (692±220SFCs/106splenocytes) and pSV1.0TRIVN mixed with CTB (734±240SFCs/106splenocytes). Though TRIVN-CTA and TRIVN-CTB fusion vaccines mounted comparable level of total IFN-g+ T-cell responses, only TRIVN-CTB elicits significantly T-cell responses against Tat, which is a subdominant component in the fusion immunogen. No significant differences were observed among groups inoculated with TRIVN alone or adjuvanted by CTA/CTB subunit proteins. Conclusion CTA and CTB could serve as potent adjuvants for DNA vaccine in immunogen-CTA/CTB fusion modality. Compared with CTA, CTB may enhance T-cell responses against subdominant epitopes in the immunogen and broaden the T-cell immune responses.

Hemokinin-1 As an Adjuvant Molecule Enhancing Humoral and Memory Responses to HBsAg DNA Vaccination

Incorporation of molecular adjuvants into DNA vaccines is often used to improve the induction of immune responses, but few approaches aim to specifically activate B cells for an enhanced humoral response only. Hemokinin-1 (HK-1) is a factor that activates B cells for proliferation, survival, differentiation into plasma cells, and Ab production. Therefore, we investigated if it may be used as a molecular adjuvant for DNA vaccines to elicit strong humoral and memory responses. The HK-1 coding sequence was sub-cloned as single or triple copies in-frame downstream of S2 HBsAg in the proVAX/S2 construct. Compared to mice immunized with proVAX/S2 or proVAX/S2-HK-1, proVAX/S2-3HK-1 induced a higher level of IgG production, a higher percentage of differentiated antibody-secreting plasma cells, and a higher level of T-cell proliferation. Furthermore, a higher proportion of B cells had the B220(+)CD27(+) phenotype in these groups, and specific antigen re-challenge induced a higher level of total IgG production 60 d after the last immunization, suggesting that the use of HK-1 as an adjuvant promoted immunological memory. Taken together, these results suggest that using HK-1 as an adjuvant molecule could enhance the immunogenicity of HBsAg DNA vaccines, and result in stronger humoral and memory responses. Therefore, HK-1 may lead to the development of a novel humoral-biased molecular adjuvant for an HBsAg DNA vaccine against hepatitis B infection.

C-terminal heat shock protein 70 of Mycobacterium tuberculosis as a molecular adjuvant for DNA vaccination with the porcine circovirus type 2 ORF2 (capsid) gene in mice

Mycobacterium tuberculosis heat shock protein 70 (HSP70) and the peptide binding C-terminal portion of HSP70 (amino acids 359-610; HSP70c) exert an adjuvant effect when used in vaccines. To enhance the immunogenicity of a DNA vaccine against porcine circovirus type 2 (PCV2), recombinant plasmids encoding the PCV2 ORF2 (capsid) gene fused to full length hsp70 (pCA-TCH) or truncated C-terminal hsp70c (pCA-TCHc) were constructed. Immunisation of mice with pCA-TCHc induced higher serum immunoglobulin G antibody levels, stronger T helper 1 immune responses and lower PCV2 viral titres following challenge than immunisation with pCA-TCH or Cap plasmids only.

Human recombinant lactoferrin as a new adjuvant in specific DNA vaccination against ErbB2 (75.2)

Abstract Lactoferrin triggers several innate immunity mechanisms. Repeated oral administrations of a recombinant human lactoferrin (Talactoferrin, TLF, Agennix, Houston, TX) hamper the growth of both transplantable tumors and autochthonous mammary carcinomas arising in BALB/c mice transgenic for the rat ErbB2 oncogene (BALB-neuT mice). To evaluate the mechanisms that delay the tumor growth and enhance the immune response elicited by a DNA vaccine against rat ErbB2 oncogene, BALB-neuT mice received by gavage PBS or TLF (1 g/kg) once daily, 5 days per week, 3 weeks on and 1 week rest for seven cycles. Following oral TLF we observed: decrease of serum IL4 and IL5; increase of serum IL12 and IFNγ; decrease of immature myeloid cells in tumor draining lymph nodes. When BALB-neuT mice receiving oral TLF were vaccinated with 50 µg of DNA plasmid coding for the extracellular and transmembrane domain of rat ErbB2 we found: extended tumor-free survivals; increased overall survivals; decrease in the number of tumors/mouse; higher titers of vaccine-induced anti-ErbB2 antibodies; similar cytotoxicity activities against ErbB2+ tumors. These findings suggest that TLF is an interesting new adjuvant in DNA vaccination against ErbB2.

454. Pushing the Limits of Expression: Highly Optimized Next Generation Molecular Adjuvants for Mucosa-Targeting DNA Vaccines

454. Pushing the Limits of Expression: Highly Optimized Next Generation Molecular Adjuvants for Mucosa-Targeting DNA Vaccines

Surface-Engineered Gold Nanorods: Promising DNA Vaccine Adjuvant for HIV-1 Treatment

With the intense international response to the AIDS pandemic, HIV vaccines have been extensively investigated but have failed due to issues of safety or efficacy in humans. Adjuvants for HIV/AIDS vaccines are under intense research but a rational design approach is still lacking. Nanomaterials represent an obvious opportunity in this field due to their unique physicochemical properties. Gold nanostructures are being actively studied as a promising and versatile platform for biomedical application. Herein, we report novel surface-engineered gold nanorods (NRs) used as promising DNA vaccine adjuvant for HIV treatment. We have exploited the effects of surface chemistry on the adjuvant activity of the gold nanorod by placing three kinds of molecules, that is, cetyltrimethylammonium bromide (CTAB), poly(diallydimethylammonium chloride) (PDDAC), and polyethyleneimine (PEI) on the surface of the nanorod. These PDDAC- or PEI-modified Au NRs can significantly promote cellular and humoral immunity as well as T cell proliferation through activating antigen-presenting cells if compared to naked HIV-1 Env plasmid DNA treatment in vivo. These findings have shed light on the rational design of low-toxic nanomaterials as a versatile platform for vaccine nanoadjuvants/delivery systems.

Soluble multi-trimeric TNF superfamily ligand adjuvants enhance immune responses to a HIV-1 Gag DNA vaccine

DNA vaccines remain an important component of HIV vaccination strategies, typically as part of a prime/boost vaccination strategy with viral vector or protein boost. A number of DNA prime/viral vector boost vaccines are currently being evaluated for both preclinical studies and in Phase I and Phase II clinical trials. These vaccines would benefit from molecular adjuvants that increase correlates of immunity during the DNA prime. While HIV vaccine immune correlates are still not well defined, there are a number of immune assays that have been shown to correlate with protection from viral challenge including CD8+ T cell avidity, antigen-specific proliferation, and polyfunctional cytokine secretion. Recombinant DNA vaccine adjuvants composed of a fusion between Surfactant Protein D (SP-D) and either CD40 Ligand (CD40L) or GITR Ligand (GITRL) were previously shown to enhance HIV-1 Gag DNA vaccines. Here we show that similar fusion constructs composed of the TNF superfamily ligands (TNFSFL) 4-1BBL, OX40L, RANKL, LIGHT, CD70, and BAFF can also enhanced immune responses to a HIV-1 Gag DNA vaccine. BALB/c mice were vaccinated intramuscularly with plasmids expressing secreted Gag and SP-D-TNFSFL fusions. Initially, mice were analyzed 2 weeks or 7 weeks following vaccination to evaluate the relative efficacy of each SP-D-TNFSFL construct. All SP-D-TNFSFL constructs enhanced at least one Gag-specific immune response compared to the parent vaccine. Importantly, the constructs SP-D-4-1BBL, SP-D-OX40L, and SP-D-LIGHT enhanced CD8+ T cell avidity and CD8+/CD4+ T cell proliferation 7 weeks post vaccination. These avidity and proliferation data suggest that 4-1BBL, OX40L, and LIGHT fusion constructs may be particularly effective as vaccine adjuvants. Constructs SP-D-OX40L, SP-D-LIGHT, and SP-D-BAFF enhanced Gag-specific IL-2 secretion in memory T cells, suggesting these adjuvants can increase the number of self-renewing Gag-specific CD8+ and/or CD4+ T cells. Finally adjuvants SP-D-OX40L and SP-D-CD70 increased T(H)1 (IgG2a) but not T(H)2 (IgG1) antibody responses in the vaccinated animals. Surprisingly, the B cell-activating protein BAFF did not enhance anti-Gag antibody responses when given as an SP-D fusion adjuvant, but nonetheless enhanced CD4+ and CD8+ T cell responses. We present evidence that various SP-D-TNFSFL fusion constructs can enhance immune responses following DNA vaccination with HIV-1 Gag expression plasmid. These data support the continued evaluation of SP-D-TNFSFL fusion proteins as molecular adjuvants for DNA and/or viral vector vaccines. Constructs of particular interest included SP-D-OX40L, SP-D-4-1BBL, SP-D-LIGHT, and SP-D-CD70. SP-D-BAFF was surprisingly effective at enhancing T cell responses, despite its inability to enhance anti-Gag antibody secretion.

Flagellin Enhances Saliva IgA Response and Protection of Anti-caries DNA Vaccine

We and others have shown that anti-caries DNA vaccines, including pGJA-P/VAX, are promising for preventing dental caries. However, challenges remain because of the low immunogenicity of DNA vaccines. In this study, we used recombinant flagellin protein derived from Salmonella (FliC) as a mucosal adjuvant for anti-caries DNA vaccine (pGJA-P/VAX) and analyzed the effects of FliC protein on the serum PAc-specific IgG and saliva PAc-specific IgA antibody responses, the colonization of Streptococcus mutans (S. mutans) on rat teeth, and the formation of caries lesions. Our results showed that FliC promoted the production of PAc-specific IgG in serum and secretory IgA (S-IgA) in saliva of rats by intranasal immunization with pGJA-P/VAX plus FliC. Furthermore, we found that enhanced PAc-specific IgA responses in saliva were associated with the inhibition of S. mutans colonization of tooth surfaces and endowed better protection with significant fewer caries lesions. In conclusion, our study demonstrates that recombinant FliC could enhance specific IgA responses in saliva and protective ability of pGJA-P/VAX, providing an effective mucosal adjuvant candidate for intranasal immunization of an anti-caries DNA vaccine.

CCL21 (SLC) improves tumor protection by a DNA vaccine in a Her2/neu mouse tumor model

Secondary lymphoid-tissue chemokine (SLC/CCL21) is a CC chemokine that is constitutively expressed in various lymphoid tissues and binds to chemokine receptor CCR7 on mature dendritic cells (DCs) and distinct T-and B-cell sub-populations. In vivo, CCL21 regulates the encounters between DC and T cells and thus is a key regulator of adaptive immune responses. We asked whether CCL21 is able to augment immunogenicity of a DNA-based vaccine against Her2/neu in a Balb/c mouse model with syngeneic Her2/neu+ tumor cells (D2F2/E2). Mice were vaccinated intramuscularly with plasmid DNA (pDNA) on day 1 and boosted on day 15; tumor challenge was performed subcutaneously on day 25. Coexpression of CCL21 and Her-2/neu resulted in induction of a TH1-polarized immune response and substantial improvement of the protective effect of the DNA vaccine. Coexpression of tumor antigen pDNA(Her2/neu) with both pDNA(GM-CSF) and pDNA(CCL21) as adjuvants led to further improvement of protection by the vaccine (70% tumor-free mice on day 35 vs 40% with either adjuvant alone vs 5-10% with tumor antigen alone). Our results show that CCL21 is a potent adjuvant for DNA vaccination, particularly in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF). Clinical use of a pDNA(Her2/neu/CCL21/GM-CSF) vaccine might be particularly promising in minimal residual Her2/neu+ breast cancer.

Cimetidine enhances the protective effect of GST DNA vaccine against Schistosoma japonicum

Cimetidine (CIM), a histamine-2-receptor antagonist, has a long history of safe use in gastric acid-mediated gastrointestinal disorders. In this study, we used CIM, as an adjuvant, with pEGFP-Sj26 GST (the recombinant plasmid containing enhanced green fluorescent protein gene and the gene encoding 26kDa glutathione S-transferase of Schistosoma japonicum) DNA vaccine to immunized mice and attempted to enhance the protective effect against S. japonicum. The results showed that the reduction rate of worm and egg burdens in the pEGFP-Sj26GST plus CIM group were 79.0% and 68.4%, respectively, significantly higher than that in pEGFP-Sj26GST alone group (27.0% and 22.5%, P<0.01). Compared with the pEGFP-Sj26GST alone group, mice immunized with pEGFP-Sj26GST plus CIM showed an elevated level of IFN-γ and IL-12 and a low level of IL-10 in splenocytes, while the levels of IL-4 and IL-5 showed no difference between the two groups. Our data also demonstrated that the percentage of CD4+CD25+ regulatory T cells (Tregs) was significantly decreased in the spleens of mice immunized with pEGFP-Sj26GST plus CIM. All these findings suggest that CIM as a potential schistosome DNA vaccine adjuvant can enhance the protective effect of pEGFP-Sj26GST vaccine.

The virus-induced signaling adaptor molecule enhances DNA-raised immune protection against H5N1 influenza virus infection in mice

As an adaptor molecule in the retinoic acid-inducible gene-I (RIG-I) signaling pathway, the virus-induced signaling adaptor (VISA) molecule activates NF-κB and IRF3 and thereby leads to the production of type I interferons (IFNs). To explore the potential of VISA as a genetic adjuvant for DNA vaccines, a eukaryotic expression plasmid, pVISA, was generated by cloning the VISA gene into the pVAX1vector. For comparison, the pTRIF plasmid was similarly constructed, encoding the known genetic adjuvant TRIF (TIR-domain-containing adapter-inducing interferon-β), an adapter in the Toll-like receptor (TLR) signaling pathway. Mice were immunized with the chimeric DNA vaccine pHA/NP 147–155, which encodes the HA (hemagglutinin) fused with NP (nucleoprotein) CTL epitope (NP 147–155) of H5N1 influenza virus, either alone or in combination with pVISA or pTRIF. Antigen-specific immune responses were examined in immunized mice. Our results demonstrate that co-immunization of the pHA/NP 147–155 plasmid with the VISA adjuvant augmented DNA-raised cellular immune responses and provided protection against H5N1 influenza virus challenge in mice. In addition, our data suggest that VISA acts as a stronger adjuvant for DNA immunization than TRIF. We conclude that co-inoculation with a vector expressing the adaptor molecule VISA enhanced the protective immunity against H5N1 infection induced by pHA/NP 147–155 and that VISA could be developed as a novel genetic adjuvant for DNA vaccines.

DAI (DLM-1/ZBP1) as a Genetic Adjuvant for DNA Vaccines That Promotes Effective Antitumor CTL Immunity

DNA vaccination is an attractive approach to induce antigen-specific cytotoxic CD8(+) T lymphocytes (CTLs), which can mediate protective antitumor immunity. The potency of DNA vaccines encoding weakly immunogenic tumor-associated antigens (TAAs) can be enhanced by codelivering gene-encoded adjuvants. Pattern recognition receptors (PRRs) that sense intracellular DNA could potentially be used to harness intrinsic immune-stimulating properties of plasmid DNA vaccines. Consequently, the cytosolic DNA sensor, DNA-dependent activator of interferon (IFN) regulatory factors (DAI), was used as a genetic adjuvant. In vivo electroporation (EP) of mice with a DAI-encoding plasmid (pDAI) promoted transcription of genes encoding type I IFNs, proinflammatory cytokines, and costimulatory molecules. Coimmunization with pDAI and antigen-encoding plasmids enhanced in vivo antigen-specific proliferation, and induction of effector and memory CTLs. Moreover, codelivery of pDAI effectively promoted CTL and CD4(+) Th1 responses to the TAA survivin. The DAI-enhanced CTL induction required nuclear factor κB (NF-κB) activation and type I IFN signaling, but did not involve the IFN regulatory factor 3 (IRF3). Codelivery of pDAI also increased CTL responses to the melanoma-associated antigen tyrosinase-related protein-2 (TRP2), enhanced tumor rejection and conferred long-term protection against B16 melanoma challenge. This study constitutes "proof-of-principle" validating the use of intracellular PRRs as genetic adjuvants to enhance DNA vaccine potency.

Evaluation of a DNA vaccine candidate expressing prM-E-NS1 antigens of dengue virus serotype 1 with or without granulocyte-macrophage colony-stimulating factor (GM-CSF) in immunogenicity and protection

Dengue is one of the most important mosquito-borne viral diseases. In past years, although considerable effort has been put into the development of a vaccine, there is currently no licensed dengue vaccine. In this study, we constructed DNA vaccines that carried the prM-E-NS1 genes of dengue virus serotype 1 (DV1) with or without the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, an attractive DNA vaccine adjuvant. Immunization with the plasmid pCAG-DV1/E/NS1, which expresses viral prM-E-NS1, or the bicistronic plasmid pCAG-DV1-GM, which co-expresses viral prM-E-NS1 and GM-CSF, resulted in long-term IgG response, high levels of splenocyte-secreted interferon-γ and interleukin-2, strong cytotoxic T lymphocyte activity and sufficient protection in the DV1-challenged mice. This suggested that both humoral and cellular immune responses were induced by the immunizations and that they played important roles in protection against the DV1 challenge. Interestingly, the magnitude, quality and protective capacity of the immune responses induced by immunization with pCAG-DV1/E/NS1 or pCAG-DV1-GM seemed stronger than those induced by pCAG-DV1/E (expressing viral prM-E alone). Taken together, we demonstrated that prM/E plus NS1 would be a suitable solution for the development of a DNA vaccine against DV.

In vitro analysis of the factors contributing to the antiviral state induced by a plasmid encoding the viral haemorrhagic septicaemia virus glycoprotein G in transfected trout cells

We have found out that transfection of the RTG-2 cell line with the viral haemorrhagic septicaemia virus (VHSV) glycoprotein G (GVHSV)-coding plasmid induces an anti-VHSV state, similar to that induced by poly I:C. Taking the advantage of the constitutive expression of toll-like receptor 9 gene (tlr9) in RTG-2 cells, we have investigated whether this antiviral state was induced by the cytosine-phosphodiester-guanine (CpG) motifs present in the plasmid DNA, by the endogenous expression of GVHSV protein or by both elements. For that, we have analysed the expression profile of the rainbow trout tlr9 and several genes related to TLR9-mediated immune response in the absence or presence of a lysosomotropic drug that specifically blocks TLR9–CpG DNA interaction. The results suggested that the high levels of cell protection conferred by a plasmid encoding GVHSV gene are due to GVHSV rather than to the CpG motifs within plasmid DNA. Therefore, plasmid DNA might not play a key role in the immune response elicited by DNA vaccines or perhaps other receptors instead TLR9 could be implicated in CpG motifs recognition and signalling. In addition, since RTG-2 cells express tlr9 gene, this cell line could be a good tool for screening TLR9 agonists, such as the immunomodulatory oligonucleotides (IMOs), as fish DNA vaccine adjuvants.