MTDH/AEG-1-based DNA vaccine suppresses lung metastasis and enhances chemosensitivity to doxorubicin in breast cancer

The gene MTDH/AEG-1 is overexpressed in more than 40% of breast cancer patients, and it is associated with poor clinical outcomes. Previous studies have indicated that MTDH/AEG-1 could promote metastatic lung-seeding and enhance chemoresistance. Therefore, MTDH/AEG-1 could be a candidate target against breast cancer lung metastasis. We demonstrated that MTDH/AEG-1-based DNA vaccine, delivered by attenuated Salmonella typhimurium, could evoke strong CD8⁺ cytotoxic-T-cell mediated immune responses against breast cancer. This vaccine showed anti-tumor growth and metastasis efficacy in a prophylactic setting. Importantly, in a therapeutic model, MTDH/AEG-1 vaccine was proved to increase chemosensitivity to doxorubicin and inhibit breast cancer lung metastasis. This vaccine could also prolong the life span of tumor-bearing mice without significant side effects in vivo. These results suggested that this novel DNA vaccine was effective in the inhibition of breast cancer growth and metastasis, and this vaccine in combination with chemotherapies offered new strategies for the clinical therapeutics of breast cancer metastasis.     

Intraperitoneal Administration of a Tumor-Associated Antigen SART3, CD40L,and GM-CSF Gene-Loaded Polyplex Micelle Elicits a Vaccine Effect in Mouse Tumor Models

Polyplex micelles have demonstrated biocompatibility and achieve efficient gene transfection in vivo. Here, we investigated a polyplex micelle encapsulating genes encoding the tumor-associated antigen squamous cell carcinoma antigen recognized by T cells-3 (SART3), adjuvant CD40L, and granulocyte macrophage colony-stimulating factor (GM-CSF) as a DNA vaccine platform in mouse tumor models with different types of major histocompatibility antigen complex (MHC). Intraperitoneally administrated polyplex micelles were predominantly found in the lymph nodes, spleen, and liver. Compared with mock controls, the triple gene vaccine significantly prolonged the survival of mice harboring peritoneal dissemination of CT26 colorectal cancer cells, of which long-term surviving mice showed complete rejection when re-challenged with CT26 tumors. Moreover, the DNA vaccine inhibited the growth and metastasis of subcutaneous CT26 and Lewis lung tumors in BALB/c and C57BL/6 mice, respectively, which represent different MHC haplotypes. The DNA vaccine highly stimulated both cytotoxic T lymphocyte and natural killer cell activities, and increased the infiltration of CD11c⁺ DCs and CD4⁺/CD8a⁺ T cells into tumors. Depletion of CD4⁺ or CD8a⁺ T cells by neutralizing antibodies deteriorated the anti-tumor efficacy of the DNA vaccine. In conclusion, a SART3/CD40L+GM-CSF gene-loaded polyplex micelle can be applied as a novel vaccine platform to elicit tumor rejection immunity regardless of the recipient MHC haplotype.     

DNA fusion-gene vaccination in patients with prostate cancer induces high-frequency CD8+ T-cell responses and increases PSA doubling time

We report on the immunogenicity and clinical effects in a phase I/II dose escalation trial of a DNA fusion vaccine in patients with prostate cancer. The vaccine encodes a domain (DOM) from fragment C of tetanus toxin linked to an HLA-A2-binding epitope from prostate-specific membrane antigen (PSMA), PSMA_27–35. We evaluated the effect of intramuscular vaccination without or with electroporation (EP) on vaccine potency. Thirty-two HLA-A2⁺ patients were vaccinated and monitored for immune and clinical responses for a follow-up period of 72 weeks. At week 24, cross-over to the immunologically more effective delivery modality was permitted; this was shown to be with EP based on early antibody data, and subsequently, 13/15 patients crossed to the +EP arm. Thirty-two HLA-A2^? control patients were assessed for time to next treatment and overall survival. Vaccination was safe and well tolerated. The vaccine induced DOM-specific CD4⁺ and PSMA₂₇-specific CD8⁺ T cells, which were detectable at significant levels above baseline at the end of the study (p = 0.0223 and p = 0.00248, respectively). Of 30 patients, 29 had a measurable CD4⁺ T-cell response and PSMA₂₇-specific CD8⁺ T cells were detected in 16/30 patients, with or without EP. At week 24, before cross-over, both delivery methods led to increased CD4⁺ and CD8⁺ vaccine-specific T cells with a trend to a greater effect with EP. PSA doubling time increased significantly from 11.97 months pre-treatment to 16.82 months over the 72-week follow-up (p = 0.0417), with no clear differential effect of EP. The high frequency of immunological responses to DOM-PSMA₂₇ vaccination and the clinical effects are sufficiently promising to warrant further, randomized testing.     

Molecular studies of an fi+ plasmid from strains of Salmonella typhimurium

Summary Plasmid DNA has been isolated from five fi ⁺ strains of Salmonella typhimurium of independent origin, including type 36 and LT2. The mean contour length of the plasmids was between 27.3 and 29.3 m. A variant line of S. typhimurium type 36 which was fi ^- yeilded no plasmid DNA. These results support the hypothesis that the fi ⁺ property of S. typhimurium is coded by a plasmid. In S. typhimurium 36 this plasmid, designated MP10₃₆, also appears to code for restriction of non-donor-specific phages. Molecular studies indicate that superinfection of S. typhimurium 36 with the kanamycin resistance determinant K, which results in loss of the fi ⁺ property, is correlated with loss of MP10₃₆. Reassociation experiments demonstrate a high degree of homology between the DNA of all five S. typhimurium plasmids, and between MP10₃₆ and K. MP10₃₆ has some homology with F and F-like R factors, but not with plasmids of other compatibility groups. A recombinant between an ampicillin resistance determinant and MP10₃₆ is autotransferable at low frequency. The significance of these findings is discussed.     

Antigen-specific CD8+ T cells induced by the ubiquitin fusion degradation pathway

We have developed a DNA vaccine encoding a fusion protein of ubiquitin (Ub) and target proteins at the N-terminus for effective induction of antigen-specific CD8⁺ T cells. A series of expression plasmids encoding a model antigen, ovalbumin (OVA), fused with mutated Ub, was constructed. Western blotting analyses using COS7 cells transfected with these plasmids revealed that there were three types of amino acid causing different binding capacities between Ub and OVA. Natural Ub with a C-terminal glycine readily dissociated from OVA; on the other hand, artificially mutated Ub, the C-terminal amino acid of which had been exchanged to valine or arginine, stably united with the polypeptide, while Ub with a C-terminal alanine partially dissociated. The ability of DNA vaccination to induce OVA-specific CD8⁺ T cells closely correlated with the stability of Ub fusion to OVA. Our strategy could be used to optimize the effect of genetic vaccines on the induction of CD8⁺ T cells.     

DNA vaccine encoding peptide P10 against experimental paracoccidioidomycosis induces long-term protection in presence of regulatory T cells

Paracoccidioidomycosis is a granulomatous systemic mycosis endemic in Brazil and other Latin America countries. A DNA vaccine encoding the immunoprotective peptide 10 (P10) significantly reduced the fungal burden in mice when given prior to or after intratracheal challenge with Paracoccidioides brasiliensis. Presently, the generation/expansion of CD4⁺ CD44^hi memory T cells as well as Foxp3⁺ Treg cells in mice immunized with the DNA vaccine (pcDNA3-P10) before and after infection with P. brasiliensis was investigated. Memory CD4⁺ CD44^hi T cells simultaneously with Foxp3⁺ Treg cells increased in the spleens and lungs of pcDNA3-P10 immunized mice on day 0, 30, 60 and 120 postinfection. Histopathology of the lung tissue showed minimal inflammation in immunized mice compared with the unimmunized group, suggesting a role for regulatory T cells in controlling the immunopathology. The DNA vaccine shows that the repeated immunization generates memory cells and regulatory T cells that replace the initially protective pro-inflammatory T cells conferring a long term protection while preserving the integrity of the infected tissue.     

A Two-Component DNA-Prime/Protein-Boost Vaccination Strategy for Eliciting Long-Term,Protective T Cell Immunity against Trypanosoma cruzi

In this study, we evaluated the long-term efficacy of a two-component subunit vaccine against Trypanosoma cruzi infection. C57BL/6 mice were immunized with TcG2/TcG4 vaccine delivered by a DNA-prime/Protein-boost (D/P) approach and challenged with T. cruzi at 120 or 180 days post-vaccination (dpv). We examined whether vaccine-primed T cell immunity was capable of rapid expansion and intercepting the infecting T. cruzi. Our data showed that D/P vaccine elicited CD4⁺ (30-38%) and CD8⁺ (22-42%) T cells maintained an effector phenotype up to 180 dpv, and were capable of responding to antigenic stimulus or challenge infection by a rapid expansion (CD8>CD4) with type 1 cytokine (IFNγ⁺ and TFNα⁺) production and cytolytic T lymphocyte (CTL) activity. Subsequently, challenge infection at 120 or 180 dpv, resulted in 2-3-fold lower parasite burden in vaccinated mice than was noted in unvaccinated/infected mice. Co-delivery of IL-12- and GMCSF-encoding expression plasmids provided no significant benefits in enhancing the anti-parasite efficacy of the vaccine-induced T cell immunity. Booster immunization (bi) with recombinant TcG2/TcG4 proteins 3-months after primary vaccine enhanced the protective efficacy, evidenced by an enhanced expansion (1.2-2.8-fold increase) of parasite-specific, type 1 CD4⁺ and CD8⁺ T cells and a potent CTL response capable of providing significantly improved (3-4.5-fold) control of infecting T. cruzi. Further, CD8⁺T cells in vaccinated/bi mice were predominantly of central memory phenotype, and capable of responding to challenge infection 4-6-months post bi by a rapid expansion to a poly-functional effector phenotype, and providing a 1.5-2.3-fold reduction in tissue parasite replication. We conclude that the TcG2/TcG4 D/P vaccine provided long-term anti-T. cruzi T cell immunity, and bi would be an effective strategy to maintain or enhance the vaccine-induced protective immunity against T. cruzi infection and Chagas disease.     

Costimulatory Effects of an Immunodominant Parasite Antigen Paradoxically Prevent Induction of Optimal CD8 T Cell Protective Immunity

Trypanosoma cruzi infection is controlled but not eliminated by host immunity. The T. cruzi trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4⁺ and CD8⁺ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against T. cruzi challenge. We generated a new TS vaccine in which the immunodominant TS CD8⁺ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8⁺ T cell epitope enhance subdominant pathogen-specific CD8⁺ T cell responses. More specifically, CD8⁺ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8⁺ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for T. cruzi vaccine development. Of broader significance, we demonstrate that increasing breadth of T cell epitope responses induced by vaccination is not always advantageous for host immunity.     

N-glycosylation-mutated HCV envelope glycoprotein complex enhances antigen-presenting activity and cellular and neutralizing antibody responses

BackgroundThe development of an efficient vaccine and broadly cross-neutralizing antibodies of hepatitis C virus (HCV) remains a priority. The heavily glycosylated viral envelope glycoprotein E1E2 complex is a candidate vaccine antigen. Bacteria-derived unmethylated CpG DNA, a potent stimulator of immune cells, is important for vaccine research.MethodsHere, the immunogenicities of wild type (WT) E1E2, five N-glycosylation site mutated E1E2 glycoproteins, and five CpG-coupled E1E2 N-glycosylation mutated glycoproteins were analyzed in BALB/c mice by DNA vaccination using in vivo electroporation.ResultsThe E1E2 protein expression levels were examined and shown to be unaffected by these N-glycosylation mutations. We found that a CpG-coupled E1-N209D-E2-N430D DNA vaccine (named CpG-E1E2-M4) induced the highest cellular immune response compared to the WT E1E2, CpG-E1E2, and other mutants. Furthermore, the CpG-E1E2-M4 anti-serum effectively neutralized the infection of cell-cultured HCV (HCVcc, genotype 2a)- and HCV pseudo particles (HCVpp, genotypes 1 to 7) to Huh-7.5.1 hepatocytes. Additionally, CpG-E1E2-M4 enhanced the Interleukin-12 (IL-12) production and antigen-presenting activity of CD11c⁺ dendritic cells (DCs) by inducing CD4⁺ Th1 polarization and the production of perforin and granzyme B (GrB) in CD8⁺ T cells.ConclusionsAs our knowledge this is the first study revealing that the naturally poor immunogenicity of E1E2 can be enhanced by the deletion of N-glycans combined with the addition of immune activator CpG by DNA vaccination.General significanceDeletion of N-glycans can enhance viral immunogenicity. The selected CpG-E1E2-M4 mutant is a novel potential HCV DNA vaccine that elicits enhanced CD4⁺ Th1 and CD8⁺ T cell responses and neutralizing antibody production against HCV infection. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.     

Ubiquitin-fusion degradation pathway: a new strategy for inducing CD8 cells specific for mycobacterial HSP65

The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8⁺ T cells. In this study, we exploited UPS to induce CD8⁺ T cells specific for mycobacterial HSP65 (mHSP65), one of the leading vaccine candidates against infection with Mycobacterium tuberculosis. A chimeric DNA termed pU-HSP65 encoding a fusion protein between murine ubiquitin and mHSP65 was constructed, and C57BL/6 (B6) mice were immunized with the DNA using gene gun bombardment. Mice immunized with the chimeric DNA acquired potent resistance against challenge with the syngeneic B16F1 melanoma cells transfected with the mHSP65 gene (HSP65/B16F1), compared with those immunized with DNA encoding only mHSP65. Splenocytes from the former group of mice showed a higher grade of cytotoxic activity against HSP65/B16F1 cells and contained a larger number of granzyme B- or IFN-γ-producing CD8⁺ T cells compared with those from the latter group of mice.     

A Legumain-based minigene vaccine targets the tumor stroma and suppresses breast cancer growth and angiogenesis

Tumor associated macrophages (TAMs) are well known to play a very important role in tumor angiogenesis and metastasis. The suppression of TAMs in the tumor-microenvironment (TME) provides a novel strategy to inhibit tumor growth and dissemination by remodeling the tumor’s stroma. Here, we tested our hypothesis that suppression of TAMs can be achieved in syngeneic BALB/c mice with oral minigene vaccines against murine MHC class I antigen epitopes of Legumain, an asparaginyl endopeptidase and a member of the C13 family of cystine proteases which is overexpressed on TAMs in the tumor stroma. Vaccine vectors were constructed and transformed into attenuated Salmonella typhimurium (Dam ⁻ , AroA ⁻ ) for oral delivery. Groups of mice received either the expression vectors encoding the Legumain H-2D or 2K epitopes or the control empty vector by gavage. The efficacy of the minigene vaccines was determined by their ability to protect mice from lethal tumor cell challenges, the induction of a specific CTL response as well as IFN-γ release, and inhibition of tumor angiogenesis. We demonstrated that the Legumain minigene vaccine provided effective protection against tumor cell challenge by inducing a specific CD8⁺ T-cell response against Legumain⁺ TAMs in our breast tumor model. The protection, induced by this T-cell response, mediated by the Legumain Kd minigene, is also responsible for lysing D2F2 breast carcinoma cells in syngeneic BALB/c mice and for suppressing tumor angiogenesis. Importantly, in a prophylactic setting, the minigene vaccine proved to be of similar anti-tumor efficacy as a vaccine encoding the entire Legumain gene. Together, our findings establish proof of concept that a Legumain minigene vaccine provides a more flexible alternative to the whole gene vaccine, which may facilitate the future design and clinical applications of such a vaccine for cancer prevention.     

CTL induction by DNA vaccine with Toxoplasma gondii-HSP70 gene

Toxoplasma gondii-derived heat shock protein 70 (T.g.HSP70) is a tachyzoite-specific virulent molecule. The DNA vaccine with T.g.HSP70 gene targeting peripheral epidermal or dermal dendritic cells (DC) induces in vivo DC maturation and successive early Th1 polarization at the draining lymph nodes (dLN) of C57BL/6 mice. In the present study, induction of cytotoxic T lymphocytes (CTL) has been explored. The CTL specific for a syngeneic DC line, DC2.4, either transfected with T. g.HSP70 gene or pulsed with recombinant T. g.HSP70 are induced in the spleen of the vaccinated mice. This CTL lyses T. gondii-infected, but not uninfected, DC2.4. Both CD8⁺ and CD4⁺ CTL are induced by the vaccine, and Fas/Fas ligand-mediated cytolysis dominantly participates in their CTL activities. Adoptive transfer experiments reveal that the vaccine-induced CD8⁺ or CD4⁺ T cells possess a protective role for toxoplasmosis at both acute and chronic phases of infection.     

Antibodies to gp120 and PD-1 Expression on Virus-Specific CD8+ T Cells in Protection from Simian AIDS

We compared the relative efficacies against simian immunodeficiency virus (SIV) challenge of three vaccine regimens that elicited similar frequencies of SIV-specific CD4⁺ and CD8⁺ T-cell responses but differed in the level of antibody responses to the gp120 envelope protein. All macaques were primed with DNA plasmids expressing SIV gag, pol, env, and Retanef genes and were boosted with recombinant modified vaccinia Ankara virus (MVA) expressing the same genes, either once (1 × MVA) or twice (2 × MVA), or were boosted once with MVA followed by a single boost with replication-competent adenovirus (Ad) type 5 host range mutant (Ad5 h) expressing SIV gag and nef genes but not Retanef or env (1 × MVA/Ad5). While two of the vaccine regimens (1 × MVA and 1 × MVA/Ad5) protected from high levels of SIV replication only during the acute phase of infection, the 2 × MVA regimen, with the highest anti-SIV gp120 titers, protected during the acute phase and transiently during the chronic phase of infection. Mamu-A*01 macaques of this third group exhibited persistent Gag CD8⁺CM9⁺ effector memory T cells with low expression of surface Programmed death-1 (PD-1) receptor and high levels of expression of genes associated with major histocompatibility complex class I (MHC-I) and MHC-II antigen. The fact that control of SIV replication was associated with both high titers of antibodies to the SIV envelope protein and durable effector SIV-specific CD8⁺ T cells suggests the hypothesis that the presence of antibodies at the time of challenge may increase innate immune recruiting activity by enhancing antigen uptake and may result in improvement of the quality and potency of secondary SIV-specific CD8⁺ T-cell responses.     

Broad and Cross-Clade CD4+ T-Cell Responses Elicited by a DNA Vaccine Encoding Highly Conserved and Promiscuous HIV-1 M-Group Consensus Peptides

T-cell based vaccine approaches have emerged to counteract HIV-1/AIDS. Broad, polyfunctional and cytotoxic CD4⁺ T-cell responses have been associated with control of HIV-1 replication, which supports the inclusion of CD4⁺ T-cell epitopes in vaccines. A successful HIV-1 vaccine should also be designed to overcome viral genetic diversity and be able to confer immunity in a high proportion of immunized individuals from a diverse HLA-bearing population. In this study, we rationally designed a multiepitopic DNA vaccine in order to elicit broad and cross-clade CD4⁺ T-cell responses against highly conserved and promiscuous peptides from the HIV-1 M-group consensus sequence. We identified 27 conserved, multiple HLA-DR-binding peptides in the HIV-1 M-group consensus sequences of Gag, Pol, Nef, Vif, Vpr, Rev and Vpu using the TEPITOPE algorithm. The peptides bound in vitro to an average of 12 out of the 17 tested HLA-DR molecules and also to several molecules such as HLA-DP, -DQ and murine IA^b and IA^d. Sixteen out of the 27 peptides were recognized by PBMC from patients infected with different HIV-1 variants and 72% of such patients recognized at least 1 peptide. Immunization with a DNA vaccine (HIVBr27) encoding the identified peptides elicited IFN-γ secretion against 11 out of the 27 peptides in BALB/c mice; CD4⁺ and CD8⁺ T-cell proliferation was observed against 8 and 6 peptides, respectively. HIVBr27 immunization elicited cross-clade T-cell responses against several HIV-1 peptide variants. Polyfunctional CD4⁺ and CD8⁺ T cells, able to simultaneously proliferate and produce IFN-γ and TNF-α, were also observed. This vaccine concept may cope with HIV-1 genetic diversity as well as provide increased population coverage, which are desirable features for an efficacious strategy against HIV-1/AIDS.     

The intact structural form of LLO in endosomes cannot protect against listeriosis

LLO is the major immuno-dominant antigen in listeriosis and is also required for protective immunity. Two forms of LLO can be observed in endosomal membranes, a LLO intact form and a Ctsd-processed LLO_1-491 form. Endosomes obtained from resting macrophages contained only LLO intact forms, while endosomes obtained from IFN-activated macrophages contained both forms. Both types of endosomes elicited LLO_90-91/CD8⁺ and LLO_189-201/CD4⁺ specific immune responses. However, only endosomes containing the Ctsd-processed LLO_1-491 form showed significant CD4⁺ and CD8⁺ T cell responses similar to LM infected bone marrow derived macrophages and characteristic of protective Listeria immunity. Moreover, endosomes with intact LLO could not confer protection as vaccine carriers against murine listeriosis. While endosomes with Ctsd-processed LLO_1-491 form showed a moderate ability, slightly lower than high efficiency vaccine vectors as MØ infected with LM. These studies argue that all cell-free membrane vesicles might serve as valid vaccine carriers against infectious agents. Exclusively those cell-free vesicles MIIC competent for LLO processing are protective vaccines vectors since they recruit significant numbers of mature dendritic cells to the vaccination sites and contain a LLO_1-491 form that might be accessible for MHC class I and class II antigen presentation.     

Regulation of purine biosynthetic genes expression inSalmonella typhimurium IV Oc mutation site ofpurG and its function analysis

Salmonella typhimurium 5 phosphoribosylformylglycinamide (FGAR) amidotransferase encoded bypurG gene catalyzes the conversion of FGAR to formylglycinamide ribonucleotide (FGAM) in the presence of glu- tamine and ATP for thede novo purine nucleotide biosynthesis.purG gene is negatively regulated by a repressor-operator system. The O⁺ purG and O^c purG were cloned respectivelyin vivo. Restriction enzymes analysis of preliminary clones pLBG-1 (O⁺) and pLBG-2 (O^c) were carried out. The hybrid plasmids pLB1933 (O⁺) and pLB1927 (O^c) containing 5′ control region ofpurG were constructed and the DNA sequences were determined respectively, DNA sequences data showed that O^c mutation ofpurG occurred at the 3rd position of 16 bp PUR box in the 5′ control region (G→A). Gel retardation experiment indicated that the repressor bound well with O⁺ PUR box, but not with O^c PUR box. The result strongly supported the idea that PUR box is the binding region of repressor protein and the 3rd position base G of PUR box is essential for the binding function with repressor protein.     

Combination of DNA Prime – Adenovirus Boost Immunization with Entecavir Elicits Sustained Control of Chronic Hepatitis B in the Woodchuck Model

A potent therapeutic T-cell vaccine may be an alternative treatment of chronic hepatitis B virus (HBV) infection. Previously, we developed a DNA prime-adenovirus (AdV) boost vaccination protocol that could elicit strong and specific CD8⁺ T-cell responses to woodchuck hepatitis virus (WHV) core antigen (WHcAg) in mice. In the present study, we first examined whether this new prime-boost immunization could induce WHcAg-specific T-cell responses and effectively control WHV replication in the WHV-transgenic mouse model. Secondly, we evaluated the therapeutic effect of this new vaccination strategy in chronically WHV-infected woodchucks in combination with a potent antiviral treatment. Immunization of WHV-transgenic mice by DNA prime-AdV boost regimen elicited potent and functional WHcAg-specific CD8⁺ T-cell response that consequently resulted in the reduction of the WHV load below the detection limit in more than 70% of animals. The combination therapy of entecavir (ETV) treatment and DNA prime-AdV boost immunization in chronic WHV carriers resulted in WHsAg- and WHcAg-specific CD4⁺ and CD8⁺ T-cell responses, which were not detectable in ETV-only treated controls. Woodchucks receiving the combination therapy showed a prolonged suppression of WHV replication and lower WHsAg levels compared to controls. Moreover, two of four immunized carriers remained WHV negative after the end of ETV treatment and developed anti-WHs antibodies. These results demonstrate that the combined antiviral and vaccination approach efficiently elicited sustained immunological control of chronic hepadnaviral infection in woodchucks and may be a new promising therapeutic strategy in patients.     

DNA Vaccines: MHC II-Targeted Vaccine Protein Produced by Transfected Muscle Fibres Induces a Local Inflammatory Cell Infiltrate in Mice

Vaccination with naked DNA holds great promise but immunogenicity needs to be improved. DNA constructs encoding bivalent proteins that bind antigen-presenting cells (APC) for delivery of antigen have been shown to enhance T and B cell responses and protection in tumour challenge experiments. However, the mechanism for the increased potency remains to be determined. Here we have constructed DNA vaccines that express the fluorescent protein mCherry, a strategy which allowed tracking of vaccine proteins. Transfected muscle fibres in mice were visualized, and their relationship to infiltrating mononuclear cells could be determined. Interestingly, muscle fibers that produced MHC class II-specific dimeric vaccine proteins with mCherry were for weeks surrounded by a localized intense cellular infiltrate composed of CD45⁺, MHC class II⁺ and CD11b⁺ cells. Increasing numbers of eosinophils were observed among the infiltrating cells from day 7 after immunization. The local infiltrate surrounding mCherry⁺ muscle fibers was dependent on the MHC II-specificity of the vaccine proteins since the control, a non-targeted vaccine protein, failed to induce similar infiltrates. Chemokines measured on day 3 in immunized muscle indicate both a DNA effect and an electroporation effect. No influence of targeting was observed. These results contribute to our understanding for why targeted DNA vaccines have an improved immunogenicity.