Combined DNA Vaccine Research Articles

Pay more attention to the immunologically comparative evaluation of HIV-1 DNA vaccine in combination with adjuvant cytokines: a long way to go

To the Editor: A recent article entitled “HIV-1 DNA vaccine with adjuvant cytokines induces specific immune responses against HIV-1 infection in mice” was published in the Chinese Medical Journal.1 The authors descriptively analyzed the adjuvant potential of an interleukin-12 (IL-12), interleukin-18 (IL-18), regulated on activation of normal T-cell expressed and secreted factor (RANTES), macrophage inflammatory protein 1 alpha (MIP-1α) or stromal cell derived factor 1 (SDF-1) plasmid when coimmunized with an HIV Gag gene-based DNA vaccine. Consequently the authors believed that IL-12, IL-18, or SDF-1 plasmid DNA potentially serves as immune adjuvants in combination with therapeutic HIV-1 DNA vaccine. The RANTES, MIP-1α plasmid DNA potentially serves as immune adjuvants in combination with HIV-1 prophylactic vaccine. Although the experiment allows us to consider such a coinoculation strategy as one of the means against HIV-1 infection, some special attentions should be highlighted. First, the authors did not set a saline control group, while female BALB/c mice were grouped. If the control were added, the analysis might be more convincing. After all, the eukaryotic expression vector might have the ocogenic potential or other detrimental effects. Second, the authors stated that the Gag gene was cloned into the pCI-neo mammalian expression vector. Some foreign genes used were cloned into other different vectors such as pVAX1. However, it is not clear which vectors were used for RANTES, MIP-1α, and SDF-1, although the authors mentioned that these recombinant plasmids were generous gifts from other researcher. In fact, the author might have ignored the immune discrepancy caused by the expression efficacy of different vectors, which may result in the uncertainty of the conclusion. Evidently, to construct and compare the expression ability of different eukaryotic expression vectors is a ceaseless work in vaccinology and other related fields. Third, although the authors have employed ELISA to detect the antibody production and the level of IFN-γ as well as performed the lymphoproliferation assays, some more direct analyses including immunofluorescence and Western blot may be added in order to confirm the expression of the genes of interest. Alternatively, immunopathology is also a suitable complimentary to this kind of study. Fourth, the usage of the female BALB/c mice only as animal models for HIV study is not very optimal, compared with that of primates. If other animals could not be adopted because of the limitation of experimental condition, both male and female mice should be used to get a more objective evaluation. Last but not least, the whole immune system is a very complicated network. Although the published article did a contribution to the comparative evaluation of the immune efficacy of HIV-1 DNA vaccine with adjuvant cytokines against HIV-1 infection in mice, currently however, it is difficult to predict whether such a contribution can ultimately clear organism from HIV or induce effective immunoreaction regardless of whether some cytokines and other beneficial components are combined.2 HIV is posing a threat against our life and the current knowledge with respect to HIV is rather limited. Both humoral and cellular immune responses will be responsible for the elimination of HIV infection. To date studies on protective effect of DNA- mediated immunization with a combination of various adjuvants against infection of different pathogens have received extensive attentions both nationally and internationally.3–6 More endeavors are needed for elucidating the immunologic mechanisms of DNA direct injection, although no specialist in the field of immunology seemed able to agree with another.

A Combined DNA Vaccine-Prime, BCG-Boost Strategy Results in Better Protection Against Mycobacterium bovis Challenge

In this study, we demonstrated that calves vaccinated with a combined DNA vaccine encoding Ag85B, MPT- 64, and MPT-83 antigens from the Mycobacterium tuberculosis for the priming and subsequently boosting with BCG prior to experimental challenge with virulent Mycobacterium bovis (M. bovis) resulted in improved immune responses over immunizing. Vaccination with the combined DNA/BCG induced higher levels of antigen- specific gamma interferon (IFN-gamma) in whole-blood cultures 4 weeks after final vaccination and the level of antigen-specific IFN-gamma in response to Ag85, MPT-64, and MPT-83 were still higher 4 weeks after challenge when compared to the combined DNA group. There was a significant bias toward induction of CD4+ T cells rather than CD8+ T cells responses, and the mean percentage of CD4+ T cells was increased about 2.6-fold in peripheral blood mononuclear cells (PBMC) cultures in DNA prime-BCG boost vaccination when compared to the nonvaccinated group. In addition, DNA prime-BCG boost vaccination resulted in stronger humoral immune responses, and the levels of the specific antibodies to three antigens were increased two- to 32- fold when compared to the combined DNA group. Vaccination with the combined DNA/BCG induced a high level of protection against an intratracheal challenge with virulent M. bovis, based on a significant enhancement of six pathological and microbiological parameters of protection compared to the nonvaccinated group. Finally, the combined DNA/BCG increased the protective efficacy by more than 10-100-fold as measured by reduced CFU counts in the lungs from calves challenged with M. bovis compared to the combined DNA and BCG groups. These results suggest that use of the prime-boost strategy offers better protection against bovine tuberculosis than does the combined DNA vaccines and BCG.

Improved efficacy of DNA vaccination against breast cancer by boosting with the repeat beta-hCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65

Studies have demonstrated that active-specific immunotherapy has potential for controlling mammary tumor progression. Human chorionic gonadotropin (hCG) is expressed and extremely sensitive, easily detectable and highly correlated with breast cancer. We developed a gene vaccine using a plasmid vector to deliver the six copies of 10-amino acid residues of β-hCG 109–118 and β hCG C-terminal 37-amino acid (CTP37). BALB/c female mice were immunized with a combination of pCR-HBc-X6-βhCGCTP37 DNA vaccine and HSP-X6-βhCGCTP37 protein vaccine. pCR-HBc-X6-βhCGCTP37 DNA vaccine were injected intramuscularly three times, on days −46,−25 and −11 and HSP-X6-βhCGCTP37 protein were applied two times, 21 and 14 days before tumor cell challenge. We assessed a combined DNA and protein vaccine for its effect of against murine EMT6 mammary tumor cells. In this study, animals vaccinated DNA vaccination boosting with the repeat β-hCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65 induced higher avidity antibodies and effectively inhibited the growth of tumor, compared with treatment using DNA alone or BCG priming HSP-X6-βhCGCTP37 protein boosting. The data presented demonstrate that improve immunogenicity of DNA vaccination by boosting with the repeat β-hCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65, which should prove useful in the development of new DNA vaccine against growth factors for cancer immunotherapy.

Comparative study of macrophage response in mice after DNA immunization and infection with herpes simplex virus type 1

Functional activity of macrophages and intensity of T cell immune response in mice were studied after intravaginal and intraperitoneal infection with herpes simplex virus type 1 and DNA vaccination in combination with adjuvant treatment (recombinant granulocyte-macrophage colony-stimulating factor and glucosaminylmuramyl dipeptide). DNA vaccination induced a virus-specific T cell immune response with no macrophagic inflammatory reaction. Infection with herpes simplex virus type 1 was accompanied by sustained inflammation, but not by the T cell immune response.

Coadministration of Interleukin 2 Plasmid DNA with Combined DNA Vaccines Significantly Enhances the Protective Efficacy AgainstMycobacterium tuberculosis

Coadministration of interleukin 2(IL-2) plasmid DNA with combined DNA vaccines enhanced Th1-type cellular responses by producing higher amounts of IFN-gamma with a higher ratio of antigen-specific IgG2a/IgG1. The IFN-gamma specific for Ag85B, MPT64, and MPT83 in this group was 415, 267, and 255 U/ml, respectively, and was 1.6-, 1.8-, and 2.5-fold higher than that of the same vaccine without adding IL-2. The IgG2a/IgG1 ratio for Ag85B, MPT64, and MPT83 was 4, 8, and 4, respectively, upon addition of the genetic adjuvant in the DNA vaccine, which was four times higher for every antigen when IL-2 was not included. Fluorescence activated cell sorter (FACS) analysis showed that, in the presence of IL-2, CD8+ and CD4+ T cells increased significantly, whereas in the absence of the genetic adjuvant, only a mild increase was observed for CD8+ T cells compared to the vector DNA-treated group. Bacterial CFU was reduced to less than 1/100 in the lung and to about 1/10 in the spleen relative to the same combined DNA vaccine without IL-2. The lungs of this group of mice showed much less damage due to an influx of epithelioid macrophages and less lymphocytes. RT-PCR showed that antigen genes could be detected in more organs and for a longer period of time when treated with combined DNA vaccine formulated in IL-2. We suggest that IL-2 enhanced the immunigencity and protective efficacy in immunized mice by improving the Th1-type response and also by prolonging the antigen gene expression in different organs.

Immunogenicity of combination DNA vaccines for Rift Valley fever virus, tick-borne encephalitis virus, Hantaan virus, and Crimean Congo hemorrhagic fever virus

DNA vaccines for Rift Valley fever virus (RVFV), Crimean Congo hemorrhagic fever virus (CCHFV), tick-borne encephalitis virus (TBEV), and Hantaan virus (HTNV), were tested in mice alone or in various combinations. The bunyavirus vaccines (RVFV, CCHFV, and HTNV) expressed Gn and Gc genes, and the flavivirus vaccine (TBEV) expressed the preM and E genes. All vaccines were delivered by gene gun. The TBEV DNA vaccine and the RVFV DNA vaccine elicited similar levels of antibodies and protected mice from challenge when delivered alone or in combination with other DNAs. Although in general, the HTNV and CCHFV DNA vaccines were not very immunogenic in mice, there were no major differences in performance when given alone or in combination with the other vaccines.

Combined DNA vaccine encapsulated in microspheres enhanced protection efficacy against Mycobacterium tuberculosis infection of mice

In a study to develop novel vaccination strategies against tuberculosis, we encapsulated DNA encoding Ag85B, MPT-64 and MPT-83 antigens mixed with dimethyldioctyldecyl ammonium bromide (DDA) into biodegradable poly( dl-lactide-co-glycolide, PLGA) microspheres. Scanning electron microscopy (SEM) analysis demonstrated a uniform microsphere population with a mean diameter of <5 μm. Using RT-PCR we were able to demonstrate antigen gene expression in selected tissue. Moreover, in mice injected with PLGA encapsulated DNA, the levels of expression appeared to be higher comparing to those injected with non-encapsulated DNA. Also, C57BL/6 mice immunized with a single dose of PLGA encapsulated DNA produced increased levels of IFN-γ in the supernatant of spleen cells when cultured in the presence of the recombinant antigens. High levels of specific IgG antibody against the three antigens were also observed. In vaccine/challenge experiments, mice receiving a single dose of PLGA encapsulated DNA were protected against Mycobacterium tuberculosis challenge at levels comparable to groups of mice immunized with three doses of non-encapsulated DNA vaccine or with Mycobacterium bovis BCG.

Combined DNA vaccines formulated either in DDA or in saline protect cattle from Mycobacterium bovis infection

We tested the effectiveness of combined DNA vaccines encoding antigens Ag85B, MPT64 and MPT83 from Mycobacterium tuberculosis on cattle. Our results showed that calves treated with combined DNA vaccines in the presence of dimethyldioctyldecyl ammonium bromide (DDA) or saline elicited a strong gamma interferon (IFN-γ) response 1 or 2 months after the third vaccination. All three antigens induced substantial levels of IFN-γ production 1 month after the bacterial challenge, when the BCG-driven IFN-γ levels dropped to less than one third of their peak values. Animals receiving combined DNA vaccines produced highest amounts of IgG antibody titer 2 months after the third vaccination. Steady state low IgG levels were found 2 months after bacterial challenge. A few small lung and lymph node lesions were detected in 1/5 animals treated with combined DNA vaccines, whereas 3/5 of BCG-treated and 5/5 of vector-control calves showed larger and significantly more lesions. About 70- to 100-fold fewer bacteria were found in the lungs and lymph nodes of combined DNA vaccine-treated animals compared to those of the control group. Histopathological analyses showed that vaccinated calves possessed substantially improved post-infection lung and lymph node pathology relative to the controls. Our data indicate that combined DNA vaccines may be used in cattle to combat bovine tuberculosis.

A Limited Autoimmunity to p185neu Elicited by DNA and Allogeneic Cell Vaccine Hampers the Progression of Preneoplastic Lesions in HER-2/NEU Transgenic Mice

Prevention of the progression of precancerous lesions by vaccines is virtually uncharted territory. Their potential, however, is being assessed in transgenic mice which develop autochthonous tumors with defined stages of progression. In this paper we show that the DNA micro-array technology significantly helps assessment of the preventive efficacy of a combined DNA and cell vaccine. All female rat Her-2/neu transgenic BALB/c (BALB-neuT) mice develop an invasive carcinoma in each of their mammary glands within 25 weeks of age. This is elicited by the activated transforming rat Her-2/neu oncogene embedded in their genome. We have previously shown that vaccination of mice bearing multiple in situ carcinomas with DNA plasmids which code for the extracellular and transmembrane domain of rat p185neu, the product of the rat Her-2/neu oncogene, followed by a boost with rat p185neu+ allogeneic cells engineered to secrete interferon-gamma, keeps 48% of mice tumor free until week 32. We have now extended our follow-up until mice reach one year of age and show that protection vanishes as time progresses. This observation suggests that the accuracy of the results studying immunotherapy against life-threatening tumors is a function of the length of the follow-up. The application of microarrays, and the concordance of morphologic and gene expression data led us to identify antibody as the main mechanism induced by vaccination. Protection is associated with a break of tolerance and a limited autoimmunity against the endogenous mouse p185neu.

Protection against Atlantic halibut nodavirus in turbot is induced by recombinant capsid protein vaccination but not following DNA vaccination

Fish nodaviruses (betanodaviruses) are small, non-enveloped icosahedral single-stranded positive-sense RNA viruses that can cause viral encephalopathy and retinopathy (VER) in a number of cultured marine teleost species, including Atlantic halibut ( Hippoglossus hippoglossus). A recombinant protein vaccine and a DNA vaccine were produced, based on the same capsid-encoding region of the Atlantic halibut nodavirus (AHNV) genome, and tested for protection in juvenile turbot ( Scophthalmus maximus). Vaccine efficacy was demonstrated in the fish vaccinated with recombinant capsid protein but not in the DNA-vaccinated fish, despite the fact that in vivo expression of the DNA vaccine-encoded antigen was confirmed by RNA in situ hybridisation and immunohistochemistry. Combined DNA and recombinant vaccine administration did not improve the effect of the latter. Surprisingly, fish vaccinated with 50 μg recombinant protein demonstrated a threefold lower survival rate than the two groups that received 10 μg recombinant protein. Neither the recombinant protein vaccine nor the DNA vaccine induced anti-viral antibodies 9 weeks after immunisation, while antibodies reactive with the recombinant protein were detectable mainly in fish vaccinated with 50 μg recombinant protein. The study also demonstrates evidence of viral replication inside the myocytes of intramuscularly challenged fish.

Combined DNA vaccines formulated in DDA enhance protective immunity against tuberculosis.

This study evaluated the adjuvant Dimethyldioctyldecyl Ammonium Bromide (DDA) effect on the protective immunity induced by a combination of plasmids containing genes encoding antigens Ag85B, MPT-83, and ESAT-6 from Mycobacterium tuberculosis. The combined DNA vaccines in DDA resulted in significant increases in both specific IgG and splenic T-cell-derived Th1-type cytokine gamma interferon (IFN-gamma) production in response to the three purified antigens when compared to that of combined DNA vaccines in saline. Vaccines in DDA increased the protective efficacy of mice challenged with M. tuberculosis H37Rv as measured by reduced relative CFU counts in their lungs. Mice immunized with the combined DNA vaccines were shown to limit the growth of tubercle bacilli both in lungs and in spleens. Histopathological analyses showed that vaccinated mice had substantially improved postinfection lung pathology relative to the controls. We suggest that our combination of antigens together with DDA formulation may provide a new insight into tuberculosis prevention.

579. Generation of Potent Cellular Immune Responses Induced by Intradermal Delivery of Plasmid DNA Coexpressing Flt3 Ligand and GM-CSF

The protective efficacy of DNA vaccine has been demonstrated in murine models against infectious diseases or tumor growth. Recently, Fms-like tyrosine kinase 3-ligand (Flt3L) has been identified as an important cytokine for the generation of professional antigen-presenting cells (APCs), particularly dendritic cells (DCs). Administration of Flt3L protein or recombinant DNA encoding Flt3L into mice results in the expansion of the DC population in vivo. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is also critical for DC survival and differentiation in vitro. Previous studies have shown that codelivery of Flt3L-encoding and GM-CSF-encoding plasmids with antigen results in increase in DC numbers in comparison with codelivery of either plasmid alone. In this study, we constructed a bicistronic plasmid vector, Flt3L-IRES-GM-CSF plasmid, coexpressing murine Flt3L lacking intracellular domain and GM-CSF by using an internal ribosome entry site (IRES). Expressions of GM-CSF and Flt3L could be detected in COS-7 cells transfected with the plasmid. The immune stimulatory activity of plasmid vector coexpressing Flt3L and GM-CSF (coexpressed DNA vaccine) in comparison with codelivery of Flt3L and GM-CSF plasmids (codelivered DNA vaccine) was studied in mice. We first immunized mice by intradermal injection of various plasmid vectors, including 40 μg Flt3L-IRES-GM-CSF plasmid, 40 μg Flt3L plasmid plus 40 μg GM-CSF plasmid, 40 μg Flt3L plasmid alone, 40 μg GM-CSF plasmid alone, or 40 μg control plasmid. Expressions of Flt3L and GM-CSF were not detected in the sera of mice receiving intradermal injection of the plasmids. Next, we showed the enhanced recruitment of DCs to the immunization site and proliferation of DCs and T cells in the spleen and lymph node. The populations of DC, CD4+, and CD8+ T cells were increased significantly by approximately 50% to 60% in mice receiving coexpressed DNA vaccine compared with those in mice receiving control plasmid. Compared to mice treated with codelivered DNA vaccine, there were 15% and 41% increases in the populations of DC and CD4+ T cells, respectively. The CD4+ but not CD4+CD25+ T cells were found to be the major group in the increased T-cell population in the spleen and lymph node. We will investigate the function of the DCs harvested from mice receiving coexpressed DNA plasmid by mixed lymphocyte reaction and antigen-specific T cell proliferation. In addition, we found that there was an increase, ranging from 20% to 40%, in the populations of MHC class II+, CD4+, and CD8+ T cells in the spleen of mice bearing syngeneic bladder tumor that received coexpressed DNA vaccine compared with those that received codelivered DNA vaccine or control plasmid. Immunization of mice with coexpressed DNA vaccine delayed bladder tumor growth. Taken together, regarding the application of Flt3L and GM-CSF, our results indicate that coexpressed DNA vaccine induces at least equal or higher immune stimulatory response compared with codelivered DNA vaccine, while only half the amount of DNA is needed. Currently, we are using the coexpressed DNA vaccine in combination with plasmid DNA encoding tumor epitopes or tumor-specific oncolytic virus for immune-gene therapy of cancer.

Multi-subtype gp160 DNA immunization induces broadly neutralizing anti-HIV antibodies.

A highly desirable feature for an human immunodeficiency virus type 1 (HIV-1) vaccine is the ability to induce broadly reactive anti-envelope antibodies that can neutralize primary HIV-1 isolates. Two immunizations with an HIV-1 envelope-encoding plasmid together with recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) resulted in high antibody titers in mice. The antibody induction was further enhanced after immunization with genes encoding HIV-1 envelopes originating from subtypes A, B and C. The sera from these animals were able to neutralize A, B and C viral isolates, whereas the sera from animals immunized solely with subtype B DNA neutralized only subtype B virus. The combined DNA vaccine gave serum antibodies with broad recognition of HIV-1 envelope epitopes as determined by peptide mapping. Cell-mediated immunity was not compromised by the increased humoral immunity. This demonstrates the ability of multiple envelope genes to induce the desired antibody response against several subtypes. Moreover, it documents the ability of rGM-CSF to enhance the potency of such a vaccine when given simultaneously. The strategy may be useful for making an HIV vaccine more potent and broadly effective against strains of different clades.

Concordant morphologic and gene expression data show that a vaccine halts HER-2/neu preneoplastic lesions

While much experimental data shows that vaccination efficiently inhibits a subsequent challenge by a transplantable tumor, its ability to inhibit the progress of autochthonous preneoplastic lesions is virtually unknown. In this article, we show that a combined DNA and cell vaccine persistently inhibits such lesions in a murine HER-2/neu mammary carcinogenesis model. At 10 weeks of age, all of the ten mammary gland samples from HER-2/neu–transgenic mice displayed foci of hyperplasia that progressed to invasive tumors. Vaccination with plasmids coding for the transmembrane and extracellular domain of rat p185neu followed by a boost with rp185neu+ allogeneic cells secreting IFN-γ kept 48% of mice tumor free. At 22 weeks, their mammary glands were indistinguishable from those of 10-week-old untreated mice. Furthermore, the transcription patterns of the two sets of glands coincided. Of the 12,000 genes analyzed, 17 were differentially expressed and related to the antibody response. The use of B cell knockout mice as well as the concordance of morphologic and gene expression data demonstrated that the Ab response is the main mechanism facilitating tumor growth arrest. This finding suggests that a new way can be found to secure the immunologic control of the progression of HER-2/neu preneoplastic lesions.

Concordant morphologic and gene expression data show that a vaccine halts HER-2/neu preneoplastic lesions.

While much experimental data shows that vaccination efficiently inhibits a subsequent challenge by a transplantable tumor, its ability to inhibit the progress of autochthonous preneoplastic lesions is virtually unknown. In this article, we show that a combined DNA and cell vaccine persistently inhibits such lesions in a murine HER-2/neu mammary carcinogenesis model. At 10 weeks of age, all of the ten mammary gland samples from HER-2/neu-transgenic mice displayed foci of hyperplasia that progressed to invasive tumors. Vaccination with plasmids coding for the transmembrane and extracellular domain of rat p185neu followed by a boost with rp185neu+ allogeneic cells secreting IFN-gamma kept 48% of mice tumor free. At 22 weeks, their mammary glands were indistinguishable from those of 10-week-old untreated mice. Furthermore, the transcription patterns of the two sets of glands coincided. Of the 12,000 genes analyzed, 17 were differentially expressed and related to the antibody response. The use of B cell knockout mice as well as the concordance of morphologic and gene expression data demonstrated that the Ab response is the main mechanism facilitating tumor growth arrest. This finding suggests that a new way can be found to secure the immunologic control of the progression of HER-2/neu preneoplastic lesions.

Treatment of bladder carcinomas using recombinant BCG DNA vaccines and electroporative gene immunotherapy.

Intravesical immunotherapy with live Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the treatment of choice for superficial bladder cancers. Nevertheless, a significant proportion of patients do not respond to this therapy, and adverse effects are common. Here, we report the cloning of recombinant mycobacterial DNA vaccines and demonstrate the ability of multicomponent and multisubunit DNA vaccines to enhance Th1-polarized cytokine-mediated responses as well as effector cell responses. Splenocytes from immunized groups of mice were restimulated in vitro and examined for cytotoxicity against murine bladder tumur (MBT-2) cells. We used four combined recombinant BCG DNA vaccines (poly-rBCG) for electroporative gene immunotherapy (EPGIT) in vivo, and found that tumor growth was significantly inhibited and mouse survival was prolonged. Increased immune cell infiltration and induction of apoptosis were noted after treatment with poly-rBCG alone, with the murine interleukin-12 (mIL-12) vaccine alone, and-most significantly-with the poly-rBCG+mIL-12 vaccine combination. Electroporation of poly-rBCG+mIL-12 resulted in complete tumor eradication in seven of eight mice (P<.01) within 28 days. Thus, EPGIT using multicomponent multisubunit BCG is highly effective in the treatment of bladder cancer. This approach presents new possibilities for the treatment of bladder cancer using recombinant BCG DNA vaccines.

Therapeutic effect of DNA vaccines combined with chemotherapy in a latent infection model after aerosol infection of mice with Mycobacterium tuberculosis.

The prevention of Mycobacterium tuberculosis (M. tuberculosis) reactivation would greatly reduce the incidence of the disease, particularly among the elderly. Here, we evaluated the efficacy of DNA vaccine in combination with a conventional TB chemotherapy on the prevention of M. tuberculosis reactivation. Mice were treated with isoniazid and pyrazinamide for 3 months from 4 weeks after aerosol infection with M. tuberculosis H37Rv. During this period of chemotherapy, DNA immunization was performed three times monthly with an antigen 85A (Ag85A) DNA or an IL-12 mutant (IL-12N220L) DNA, which is known to lead to a reduction in the secretion of the p40 subunit, but not of a bioactive IL-12p70. The reactivation of M. tuberculosis was dramatically reduced in mice treated with either Ag85A DNA (P<0.01) or IL-12N220L DNA (P<0.05) in combination with chemotherapy, compared with control mice receiving only chemotherapy. Ag85A DNA vaccine showed higher IFN-gamma responses to Ag85A protein, but a lower response to culture filtrate than IL-12N220L DNA vaccine. In addition, Ag85A DNA vaccine prevented the reactivation of M. tuberculosis more efficiently than IL-12N220L DNA vaccine, indicating that Ag85A-specific IFN-gamma response might correlate with M. tuberculosis control. This study suggests that immunotherapy using Ag85A or IL-12N220L DNA vaccine combined with conventional chemotherapy might be effective clinically for the prevention of tuberculosis reactivation and may offer a more effective cure for humans than chemotherapy alone.

Evidence for antigen production in muscles by dengue and Japanese encephalitis DNA vaccines and a relation to their immunogenicity in mice

This study demonstrated viral antigen production in muscle tissues following inoculation with DNA vaccines and examined its relation to antibody induction in mice using the flavivirus system. To achieve detectable levels of antigen production, we used a needle-free jet injector and examined 10% homogenate of quadriceps muscle for viral antigens in a sandwich enzyme-linked immunosorbent assay. We compared DNA vaccines against dengue type 1 (designated pcD1ME), dengue type 2 (pcD2ME) and Japanese encephalitis (pcJEME). The amounts of viral envelope (E) antigen contained in muscle homogenate 1, 2, 3 and 4 days following inoculation with 50μg of pcJEME were 1.1, 1.0, 0.3 and <0.1ng/ml, respectively. Muscles from pcD2ME- and pcD1ME-inoculated mice did not contain detectable levels of E antigen (<0.1ng/ml) during 4 days following inoculation. The E amounts released from Vero cells transfected with DNAs were in the order pcJEME>pcD2ME>pcD1ME. Levels of neutralizing antibody induced by two immunizations with 100μg of each DNA vaccine using needle-free or normal needle/syringe injection systems also were in the order pcJEME>pcD2ME>pcD1ME, 2–11 weeks after the first immunization. However, the difference in antibody levels among three DNA vaccines 14–18 weeks after immunization was smaller than that in the early phase of immunization. These results provide fundamental information useful for developing combination DNA vaccines, such as a dengue tetravalent DNA vaccine, which require adjustment of immunogenicity of each component.

Comparison of individual and combination DNA vaccines for B. anthracis, Ebola virus, Marburg virus and Venezuelan equine encephalitis virus

Multiagent DNA vaccines for highly pathogenic organisms offer an attractive approach for preventing naturally occurring or deliberately introduced diseases. Few animal studies have compared the feasibility of combining unrelated gene vaccines. Here, we demonstrate that DNA vaccines to four dissimilar pathogens that are known biowarfare agents, Bacillus anthracis, Ebola (EBOV), Marburg (MARV), and Venezuelan equine encephalitis virus (VEEV), can elicit protective immunity in relevant animal models. In addition, a combination of all four vaccines is shown to be equally as effective as the individual vaccines for eliciting immune responses in a single animal species. These results demonstrate for the first time the potential of combined DNA vaccines for these agents and point to a possible method of rapid development of multiagent vaccines for disparate pathogens such as those that might be encountered in a biological attack.

DNA injection in combination with electroporation: a novel method for vaccination of farmed ruminants.

Injection of plasmid DNA encoding antigens into rodents followed by electroporation improved the immune response when compared with injection without electroporation (Widera et al. J Immunol 2000;164:4635-40; Zucchelli et al. J Virol 2000;74:11598-607; Kadowaki et al. Vaccine 2000;18:2779-88). The present study describes the extension of this technology to farm animals, by injecting plasmid DNA encoding mycobacterial antigens (MPB70, Ag85B and Hsp65) into the muscles of goats and cattle using two different types of electrodes, both allowing DNA delivery at the site of electroporation. The animals were vaccinated under local anaesthesia without any observed immediate or long-term distress or discomfort, or any behavioural signs of muscle damage or pathological changes after the electroporation. DNA-injected and electroporated goats showed increased humoral response after the primary vaccination when compared with nonelectroporated animals. Improved T-cell responses following electroporation were observed in hsp65 DNA-vaccinated cattle. DNA injection with or without electroporation did not compromise the specificity of the tuberculin skin test. In conclusion, a protocol applying in vivo electroporation free of side effects to farmed ruminants was established. In addition, we show that DNA vaccination in combination with electroporation can improve the primary immune responses to the encoded antigens.