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Coxsackievirus B3 (CVB3) is an important cause of viral myocarditis with no vaccine available in clinic. Herein we constructed an exosome-based anti-CVB3 vaccine (Exo-VP1), and compared its immunogenicity and immunoprotection with our previously reported recombinant VP1 protein (rVP1) vaccine. We found that compared with the 25 μg rVP1 vaccine, Exo-VP1 vaccine containing only 2 μg VP1 protein induced much stronger CVB3-specific T cell proliferation and CTL responses (with an increase of more than 70% and 40% respectively), and elicited greater splenic Th1/CTL associated cytokines (IFN-γ, TNF-α and IL-12). Furthermore, higher IgG levels with increased neutralizing titers and avidity were also evidenced in Exo-VP1 group. Consistently, Exo-VP1 group exhibited enhanced resistance to viral myocarditis than rVP1 vaccine, reflected by reduced cardiac viral loads, improved myocardial inflammation and an increased survival rate. Collectively, we reported that Exo-VP1 might present a more potent CVB3 vaccine candidate than rVP1 vaccine., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The recurrent Coxsackievirus B3 (CVB3) infection is the most important cause of intractable myocarditis which often leads to chronic myocarditis and even dilated cardiomyopathy. Therefore, enhanced DNA vaccines capable of memory CD8 T cells are essential for long-lasting immunological protection against CVB3 infection. In this study, absent in melanoma 2 (AIM2) was used as an adjuvant to enhance the induction of memory CD8 T cells elicited by VP1 (viral capsid protein 1) vaccine. Mice were intramuscularly injected with 50 μg AIM2 plasmid and equal amount of VP1 plasmid (pAIM2/pVP1) vaccine 4 times at 2 week-intervals. We observed that the protection of pAIM2/pVP1 vaccine against CVB3 challenge was evidenced by significantly improved cardiac function, reduced myocardial injuries, and increased survival rate when compared with immunization with pVP1. Co-immunization with pAIM2/pVP1 robustly augmented T lymphocytes proliferation and CVB3-specific cytotoxic T lymphocyte responses. Importantly, 16 weeks after the last immunization, pAIM2/pVP1 co-immunization significantly enhanced the expression of Bcl-6, SOCS3, and Sca-1 which are critical for memory CD8 T cells as compared with pVP1 immunization. Notably, CD8 T cells that are likely vaccine-induced memory T cells were responsible for the protective efficacy of pAIM2/pVP1 vaccine by abolition of a CD8 T cell immune response following a lethal dose of CVB3 infection. Our results indicate that AIM2-adjuvanted vaccine could be a potential and promising approach to promote a long-lasting protection against CVB3-induced myocarditis.

Viral myocarditis is a common clinical cardiovascular disease mainly induced by coxsackievirus B3 (CVB3) with no effective therapeutic measures. Induction of efficient mucosal immune responses is very critical against CVB3-induced myocarditis. FimH is an Escherichia coli (E. coli)-derived protein, which possesses an M cell-targeting property and functions as a TLR4 agonist. In this study, we introduced the recombinant FimH protein, into our previously developed CVB3 mucosal vaccine chitosan (CS)-pVP1, aiming to provoke more efficient mucosal immune responses and immunoprotection against CVB3-induced myocarditis. Compared with the CS-pVP1 vaccine, immunization with FimH-CS-pVP1 remarkably increased the levels and neutralizing titers of CVB3-specific protective secretory IgA (sIgA), enhanced the frequency of CVB3-specific IgA-producing B cells and amplified mucosal T-cell immune responses in mesenteric lymph nodes (MLNs), although failing to significantly amplify CVB3-specific systemic immune responses. Consistently, FimH-CS-pVP1 group showed the enhanced immunoprotection against CVB3-induced myocarditis, evidenced by the indices of limited myocardial injury, reduced viral loads and enhanced survival rate. Further study showed that this enhanced immunoprotection was not only ascribed to its M cell-targeting property, which led to the enhanced mucosal antigen VP1 expression, but also associated with the mucosal adjuvant effect of FimH, which facilitated the formation of germinal centers (GCs), production of IgA-inducing factors and maturation of antigen-presenting cells (APCs). Taken together, here we developed a bi-functional mucosal vaccine FimH-CS-pVP1, which simultaneously possessed the M cell-targeting property and mucosal adjuvant ability, and we showed that FimH-CS-pVP1 could efficiently induce the higher levels of CVB3-specific protective mucosal immune responses and provide better prophylactic effects against CVB3-induced myocarditis than CS-pVP1., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Coxsackievirus B3 (CVB3) infection can cause acute myocarditis and chronic myocarditis, leading to dilated cardiomyopathy (DCM) with no effective therapeutic strategy. Therefore, we investigated the potential of absent in melanoma 2 (AIM2) to enhance the therapeutic efficacy of DNA vaccine against CVB3-induced chronic myocarditis. Mice were infected with CVB3 and then intranasally immunized with chitosan-pcDNA3.1 (mock), chitosan-pAIM2 (CS-pAIM2), chitosan-pVP1 (CS-pVP1), or chitosan-pAIM2 plus chitosan-pVP1 (CS-pAIM2/CS-pVP1) at 7, 21, and 35d. Therapeutic efficacies of various vaccines were evaluated at day 56d. Compared with CS-pVP1 immunization, CS-pAIM2/CS-pVP1 co-immunization significantly increased survival rate, improved cardiac function, as well as decreased myocardial injury and fibrosis, this result indicated that CVB3-induced chronic myocarditis was alleviated. CVB3-specific T lymphocyte proliferation and cytotoxic T lymphocyte responses of the CS-pAIM2/CS-pVP1 co-immunization group were also increased. More interestingly, CS-pAIM2/CS-pVP1 co-immunization could facilitate CVB3-specific multifunctional CD8(+) T cell induction in the intestinal mucosa, and this induction was closely correlated with myocardial scores, this result indicated that CS-pAIM2/CS-pVP1 vaccine exhibits therapeutic efficacy by enhancing multifunctional CD8(+) T cells. This study may represent a novel therapy for CVB3-induced chronic myocarditis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Efficient delivery of antigen to mucosal associated lymphoid tissue is a first and critical step for successful induction of mucosal immunity by vaccines. Considering its potential transcytotic capability, M cell has become a more and more attractive target for mucosal vaccines. In this research, we designed an M cell-targeting strategy by which mucosal delivery system chitosan (CS) was endowed with M cell-targeting ability via conjugating with a CPE30 peptide, C terminal 30 amino acids of clostridium perfringens enterotoxin (CPE), and then evaluated its immune-enhancing ability in the context of coxsackievirus B3 (CVB3)-specific mucosal vaccine consisting of CS and a plasmid encoding CVB3 predominant antigen VP1. It had shown that similar to CS-pVP1, M cell-targeting CPE30-CS-pVP1 vaccine appeared a uniform spherical shape with about 300 nm diameter and +22 mV zeta potential, and could efficiently protect DNA from DNase I digestion. Mice were orally immunized with 4 doses of CPE30-CS-pVP1 containing 50 μg pVP1 at 2-week intervals and challenged with CVB3 4 weeks after the last immunization. Compared with CS-pVP1 vaccine, CPE30-CS-pVP1 vaccine had no obvious impact on CVB3-specific serum IgG level and splenic T cell immune responses, but significantly increased specific fecal SIgA level and augmented mucosal T cell immune responses. Consequently, much milder myocarditis and lower viral load were witnessed in CPE30-CS-pVP1 immunized group. The enhanced immunogenicity and immunoprotection were associated with the M cell-targeting ability of CPE30-CS-pVP1 which improved its mucosal uptake and transcytosis. Our findings indicated that CPE30-CS-pVP1 may represent a novel prophylactic vaccine against CVB3-induced myocarditis, and this M cell-targeting strategy indeed could be applied as a promising and universal platform for mucosal vaccine development., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Development of effective anti-tuberculosis (TB) vaccines is one of the important steps to improve control of TB. Cell-mediated immune response significantly affects the control of M. tuberculosis infection. Thus, vaccines able to elicit strong cellular immune response hold special advantages against TB. In this study, three well-defined mycobacterial antigens (Rv3615c, Mtb10.4 [Rv0228], and Rv2660c) were engineered as a novel triple-antigen fusion DNA vaccine p846. The p846 vaccine consists of a high density of CD4(+) and CD8(+) T-cell epitopes. Intramuscular immunization of p846 induced robust T cells mediated immune response comparable to that of bacillus Calmette-Guérin (BCG) vaccination but more effective than that of individual antigen vaccination. After mycobacterial challenge, p846 immunization decreased bacterial burden at least 15-fold compared with individual antigen-based vaccination. Notably, the lungs of mice immunized with p846 exhibited fewer inflammatory cell infiltrates and less damage than those of control group mice. Our data demonstrate that the potential of p846 vaccine to protect against TB and the feasibility of this design strategy for further TB vaccine development.

Coxsackievirus B3 (CVB3) infection is considered as the most common cause of viral myocarditis with no available vaccine. Considering that CVB3 mainly invades through the gastrointestinal mucosa, the development of CVB3-specific mucosal vaccine, which is the most efficient way to induce mucosal immune responses, gains more and more attention. In this study, we used absent in melanoma 2 (AIM2) as a mucosal adjuvant to enhance the immunogenicity and immunoprotection of CVB3-specific chitosan-pVP1 vaccine. Mice were intranasally co-immunized with 50 μg chitosan-pAIM2 and equal amount of chitosan-pVP1 vaccine 4 times at 2 week-intervals, and then challenged with CVB3 2 weeks after the last immunization. Compared with chitosan-pVP1 vaccine immunization alone, chitosan-pAIM2 co-immunization enhanced resistance to CVB3-induced myocarditis evidenced by significantly enhanced ejection fractions from 55.40 ± 9.35 to 80.31 ± 11.35, improved myocarditis scores from 1.50 ± 0.45 to 0.30 ± 0.15, reduced viral load from 3.33 ± 0.50 to 0.50 ± 0.65, and increased survival rate from 40.0% to 75.5%. This increased immunoprotection might be attributed to the augmented level of CVB3-specific fecal SIgA with high affinity and neutralizing ability. In addition, co-immunization with chitosan-pAIM2 remarkably facilitated dendritic cells (DCs) recruitment to mesenteric lymph nodes (MLN), and promoted the expression of IgA-inducing factors (BAFF, APRIL, iNOS, RALDH1, IL-6, TGF-β), which might account for its mucosal adjuvant effect. This strategy may represent a promising prophylactic vaccine against CVB3-induced myocarditis.

Coxsackievirus B3 (CVB3), a small single-stranded RNA virus, belongs to the Picornaviridae family. Its infection is the most common cause of myocarditis, with no vaccine available. Gastrointestinal mucosa is the major entry port for CVB3; therefore, the induction of local immunity in mucosal tissues may help control initial viral infections and alleviate subsequent myocardial injury. Here we evaluated the ability of high-mobility group box 1 (HMGB1) encapsulated in chitosan particles to enhance the mucosal immune responses induced by the CVB3-specific mucosal DNA vaccine chitosan-pVP1. Mice were intranasally coimmunized with 4 doses of chitosan-pHMGB1 and chitosan-pVP1 plasmids, at 2-week intervals, and were challenged with CVB3 4 weeks after the last immunization. Compared with chitosan-pVP1 immunization alone, coimmunization with chitosan-pHMGB1 significantly (P < 0.05) enhanced CVB3-specific fecal secretory IgA levels and promoted mucosal T cell immune responses. In accordance, reduced severity of myocarditis was observed in coimmunized mice, as evidenced by significantly (P < 0.05) reduced viral loads, decreased myocardial injury, and increased survival rates. Flow cytometric analysis indicated that HMGB1 enhanced dendritic cell (DC) recruitment to mesenteric lymph nodes and promoted DC maturation, which might partly account for its mucosal adjuvant effect. This strategy may represent a promising approach to candidate vaccines against CVB3-induced myocarditis.

Induction of local (pulmonary) immunity plays a critical role in preventing dissemination of Mycobacterium tuberculosis (M. tb) during the early infection stage. To induce specific mucosal immunity, chitosan, a natural cationic polysaccharide, was employed as a mucosal gene carrier and complexed with pHSP65pep, our previously constructed multi-epitope gene vaccine, which induces splenic gamma-interferon (IFN-γ)(+) T helper cell 1 responses. The resultant chitosan-pHSP65pep was administered intranasally to BALB/c mice with four doses of 50 μg DNA followed by mycobacterial challenge 4 weeks after the final immunization. It was found that the chitosan formulation significantly induced production of secretory immunoglobulin A (P < 0.05) as determined by measuring its concentrations in lung lavage fluid and enhanced pulmonary CD4(+) and CD8(+) IFN-γ(+) T cell responses (P < 0.001) compared with naked gene vaccine. Improved protection against Mycobacterium bovis bacillus Calmette-Guérin (BCG) challenge was consistently achieved by the chitosan-DNA formulation both as the vaccine alone or in a BCG prime-vaccine boost immunization scenario. Our study shows that mucosal delivery of gene vaccine in a chitosan formulation remarkably enhances specific SIgA concentrations and mucosal IFN-γ(+) T cell response, which correlated positively with immunological protection., (© 2013 The Societies and Wiley Publishing Asia Pty Ltd.)

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a major infectious disease worldwide. Moreover, latent M. tuberculosis infection is more likely to progress to active TB and eventually leads to death when HIV infection is involved. Thus, it is urgent to develop a novel TB vaccine with immunogenicity to both M. tuberculosis and HIV. In this study, four uncharacterized T cell epitopes from MPT64, Ag85A, Ag85B, and TB10.4 antigens of M. tuberculosis were predicted, and HIV-1-derived p24, an immunodominant protein that can induce protective responses to HIV-1, was used as an immunogenic backbone. M. tuberculosis epitopes were incorporated separately into the gene backbone of p24, forming a pP24-Mtb DNA vaccine. We demonstrated that pP24-Mtb immunization induced a strong M. tuberculosis-specific cellular response as evidenced by T cell proliferation, cytotoxicity, and elevated frequency of gamma interferon (IFN-γ)-secreting T cells. Interestingly, a p24-specific cellular response and high levels of p24-specific IgG were also induced by pP24-Mtb immunization. When the protective effect was assessed after mycobacterial challenge, pP24-Mtb vaccination significantly reduced tissue bacterial loads and profoundly attenuated the mycobacterial infection-related lung inflammation and injury. Our findings demonstrated that the pP24-Mtb tuberculosis vaccine confers effective protection against mycobacterial challenge with simultaneously elicited robust immune responses to HIV-1, which may provide clues for developing novel vaccines to prevent dual infections.

Induction of potent mucosal immune response is a goal of current vaccine strategies against mucus-infectious pathogens such as Coxsackievirus B3 type (CVB3). We previously showed that administration of lymphotactin (LTN) as an adjuvant could enhance the specific immune responses against a mucosal gene vaccine, chitosan-pVP1, against CVB3. To optimize the coadministration mode of the mucosal adjuvant, we compared the mucosal immune responses induced by chitosan-DNA vaccine with different combinations of the target VP1 antigen gene and the adjuvant LTN gene. The two genes were either cloned in separate vectors or coexpressed as a fusion or bicistron protein in the same vector before encapsulation in chitosan nanoparticles. Four doses of various adjuvant-combined chitosan-DNA were intranasally administrated to mice before challenge with CVB3. The results indicated that chitosan-formulated pVP1-LTN fusion plasmid exhibited very weak improvement of CVB3-specific immune responses. Although the bicistronic coexpression of LTN with VP1 was expected to be powerful, this combination had enhanced effects on serum IgG and systemic T cell immune responses, but not on mucosal T cell immunity. Coimmunization with VP1 and LTN as separate chitosan-DNA formulation remarkably enhanced antibody and T cell immune responses both in systemic and mucosal immune compartments, leading to the most desirable preventive effect on viral myocarditis. Taken together, how the adjuvant is combined with the target antigen has a strong influence on the mucosal immune responses induced by mucosal DNA vaccines.

Vaccination is expected to make a major contribution to the goal of eliminating tuberculosis worldwide by 2050. Because the protection afforded by the currently available tuberculosis vaccine, BCG, is insufficient, new vaccine strategies are urgently needed. Protective immunity against MTB depends on generation of a Th1-type cellular immune response characterized by secretion of IFN-gamma from antigen-specific T cells. Epitope-driven vaccines are created from sub-sequences of proteins (epitopes) derived by scanning the protein sequences of pathogens and selecting epitopes with patterns of amino acids which permit binding to human MHC molecules. Guided by the crystal structure of HSP65 and its characteristics, four functional T cell epitopes elaborately elicited from ESAT-6, Ag85A, CFP-10 and Ag85B were cast into the intermediate domain of HSP65. A panel of a novel chimeric vaccine, ECANS, expressing HSP65 and combined T cell epitopes was created. Gene cloning and sequencing, DNA vaccination and humoral and cellular responses were studied. After being immunized with DNA vaccine three times, all mice injected with ECANS had specific cellular immune responses. In addition, lymphocytes obtained from the spleen of ECANS immunized mice at week eight exhibited significantly greater specific lymphocyte proliferation, IFN-gamma secretion and CTL activity than those of mice that had been immunized with BCG. DNA vaccine with ECANS can successfully induce enhanced specific cellular immune response to PPD, and further study of its protective effects against Mycobacterium tuberculosis in vivo is needed.

Antibodies to CD25 (anti-CD25) were used clinically to achieve immunosuppression in autoimmune disease, while the possibility of anti-antibodies generation influenced its efficiency. Here we reported that DNA vaccine encoding the extracellular domain of murine CD25 gene (pCD25-ECD) elicited anti-CD25 antibody production in BALB/c mice and subsequently prevents the host against ConA-induced autoimmune hepatitis. We found that serum CD25-specific antibodies were generated after vaccination with pCD25-ECD. Moreover, high levels of IL-4, IL-10 and anti-CD25 antibody were produced by splenocytes of vaccinated mice after CD25 protein restimulation in vitro. Furthermore, we demonstrated that the vaccinated mice suffered less from liver injury induced by ConA, accompanied by the reduction of pathogenic CD4+ T cells. Finally, we showed that the immunized serum could cause cytolysis of activated CD4+ T cells in vitro, depending on complements activation. Our study showed pCD25-ECD induced self anti-CD25 antibodies which executed immunosuppression in autoimmune hepatitis via antibody-complement pathway.

Epitope-based DNA vaccines designed to induce T cell responses specific for Mycobacterium tuberculosis (M. tb) are being developed as a means of addressing vaccine potency. In this study, we predicted 4 T cell epitopes from ESAT-6, Ag85A/B and CFP-10 antigens and constructed an ECANS (epitopes casted in a natural structure) DNA vaccine by inserting the epitope DNA segments separately into the gene backbone of M. tb-derived HSP65 (heat shock protein 65) carrier. The immunogenicity and protective efficacy of pECANS DNA vaccine were assessed in BALB/c mice after intramuscular immunization with 4 doses of 50 microg ECANS DNA and followed by mycobaterial challenge 4 weeks after the last immunization. Compared to plasmid encoding HSP65, pECANS DNA immunization elicited remarkably higher levels of IFN-gamma production by both CD4(+) and CD8(+) T cells, which were coupled with higher frequencies of antigen-specific T cells and higher CTL activity. Significantly enhanced levels of Th1 cytokines (IFN-gamma and IL-12) and increased serum IgG2a/IgG1 ratio were also noted, indicating a predominant Th1 immune response achieved by pECANS DNA immunization. In the consequence, a better protection against Mycobacterium bovis BCG challenge was achieved which was evidenced by reduced bacterial loads in lungs and spleens and profound attenuation of lung inflammation and injury. Our results suggested that multi-T cell-epitope based ECANS gene vaccine induced T cell response to multiple T cell epitopes and led to enhanced protection against mycobacterial challenge. This strategy might be a useful platform to design multi-T cell epitope-based vaccine against M. tb infection.

Coxsackievirus B3 (CVB3) is a gastrointestinal virus causing myocarditis in human and mice. An ideal vaccine for CVB3-myocarditis requires both humoral and cellular immunity at systemic and mucosal compartments. We described here an enhancing strategy for chitosan-pVP1 vaccine by co-immunizing with lymphotactin (LTN) gene, a T cell-attractive-chemokine, encapsulated in chitosan particle to provide more protection against CVB3. Mice were intranasally co-immunized with 4 doses of chitosan-DNA vaccines separately encapsulating VP1 and LTN plasmids by 2 week-intervals and challenged with CVB3 4 weeks after the last immunization. Compared with chitosan-pVP1 alone, co-immunization with chitosan-pLTN significantly increased high-avidity-neutralizing antibody levels in serum and in intestinal mucosa, and promoted systemic and mucosal Th1 and CD8(+)CTL immune responses. Accordingly, enhanced resistance to CVB3-myocarditis was evidenced by reduced myocardial viral load, profound subsidence of myocarditis and increased survival rate. This strategy represents a promising platform for Th1 polarization and protection against mucosal infectious pathogens.

Epitope-based vaccines designed to induce antibody responses specific for severe acute respiratory syndrome-associated coronavirus (SARS-CoV) are being developed as a means for increasing vaccine potency. In this study, we identified four B cell epitopes from the spike (S) and membrane (M) protein through bioinformatics analysis and constructed a multi-epitope DNA vaccine. Intramuscular immunization of mice with this vaccine was sufficient to induce specific prime as well as a long-term memory humoral immune response to at least two candidate epitopes, S(437-459) and M(1-20). A DNA prime-protein boost strategy greatly enhanced the antibody generation and the immune sera not only reacted with the lysates of SARS-CoV-infected Vero cells but also neutralized the cytopathic effect of SARS by 75% at 1:160 dilution. The novel immunogenic S protein peptide revealed in this study provides new target for SARS vaccine design; and our work indicated multi-epitope DNA vaccine as an effective means for eliciting polyvalent humoral immune response against SARS-CoV.

Induction of T help cell type 1 (Th1) response seems to be a prerequisite of HBV clearance. DNA vaccines have shown its potential to elicit Th1-biased immune response. However, its immunogenicity needs to be improved. Regulated upon activation normal T cell expressed and secreted (RANTES) is an inflammatory chemokine that promotes the accumulation and activation of CD4+, CD8+ T cells, and dendritic cells (DCs), which would favor antiviral immunity. In this study, the efficacy of a DNA vaccine encoding hepatitis B virus (HBV) preS2 plus S protein was enhanced through co-injection of a plasmid encoding RANTES in a BALB/c model. Co-injection of RANTES gene resulted in a moderate increase in the HBV specific humoral and cellular immune responses and a significant increase following an HBsAg booster vaccination compared to DNA encoding HBsAg alone. This enhancement was due to an enrichment of DCs in the draining lymph node and an up-regulation of DCs maturation by RANTES. More importantly, RANTES polarized the specific immunity towards a dominant Th1 profile and even converted an established Th2 response to a Th1 phenotype. Our study suggested the feasibility of using a plasmid-encoded RANTES as a modulatory Th1 adjuvant in genetic vaccination.

DNA vaccines have shown to induce strong immune response in small animals; however, its capacity of inducing robust antigen-specific immune responses in large animals is limited. In the present study, in vivo electroporation (EP) was applied and the effect of EP on humoral immune response against tuberculosis (TB) induced by DNA vaccination was tested in mice and rhesus macaques. Mice injected with 10 microg DNA encoding Ag85A and ESAT-6 followed by EP showed a reproducible humoral immunity which was equal to that obtained by using 100 microg DNA without EP. Boosting the DNA/EP treated animals with corresponding recombinant protein (50 microg of either Ag85A or ESAT-6) without adding adjuvant gave more than a 7-8-fold increase in the antibody titre but only 3-4-fold increase was found in the mice receiving 100 microg DNA without EP followed by protein boost. In concordance with the results obtained in mice, the monkeys received less DNA achieved equal high antibody responses to those induced by high dosage of DNA. Boosting the the DNA/EP treated monkeys with TB protein (500 microg of either Ag85A or ESAT-6) improved the humoral response by 7-8-fold increase in antibody titre, indicating electroporation's ability to compensate lower DNA concentration and enhance humoral immunity of TB DNA vaccines in mice and non-human primates.

Ag85A and ESAT-6 proteins of Mycobacterium tuberculosis (M.TB) are important protective antigens. The 32-kDa Ag85A is a strong immunogen in both small and large animals. However, the 6-kDa ESAT-6 has relatively low inherent immunogenicity, especially in large animals. To improve the immunogenicity of ESAT-6 in animals, we made chimeric DNA vaccines, HG856K and HG856A, by inserting the esat-6 gene into the Kpn I or Acc I endonuclease restriction site of the ag85a gene, respectively. BALB/c mice were injected intramuscularly three times with the 10-microg singular DNA vaccine (HG85 encoding for Ag85A or HG6 encoding for ESAT-6) or chimeric DNA vaccine (HG856K or HG856A) followed by electroporation (EP). Ten days after the last DNA vaccination, mice received a booster immunization intraperitoneally with 50-microg pure recombinant protein Ag85A or ESAT-6 without adjuvant. Additional groups of mice immunized with chimeric DNA vaccines were boosted with two mixed proteins (Ag85A/ESAT-6) at the same time. The results showed that the immunogenicity of M.TB ESAT-6 antigen was not improved by priming with the HG6 DNA vaccine. However, the humoral immunity against the ESAT-6 antigen was significantly increased in the mice primed with chimeric DNA vaccines, HG856K or HG856A, followed by boosting with ESAT-6 or ESAT-6/Ag85A mixed proteins.

Vitamin A deficiency diminishes Th2-mediated Ab responses. Providing Vitamin A or its active metabolites reverses this defect. All-trans retinoic acid (ATRA), an acid derivation of Vitamin A, regulates the balance of immune response induced by TR421-hCGbeta DNA vaccine. Compared to DNA vaccine alone or treatment with vehicle, significantly higher level of antibody against the protein encoded by DNA vaccine was observed in mice 6 weeks after the first immunization. The IgG2a/IgG1 ratio was lower in mice treated with ATRA. We also found that treatment with ATRA also diminishes specific cellular immune response induced by gene immunization by measuring the marker of cellular immune response. We conclude that ATRA biases the immune response to Th2 direction induced by DNA vaccine and acts as a candidate adjuvant and immunomodulatory molecule.

Epitope-based DNA vaccine is an effective and powerful approach against a variety of pathogens or tumors. In present study, we reconstructed a vector that could effectively express short B and T-cell epitope of duck/hepatitis B virus, and investigated the role of the epitope-based DNA vaccination. The pUC19 was modified by inserting the compact transient framework (CTF), including HCMV IE1 promoter, enhancer, Kozak sequence, dual stop codon and 3' terminal bovine growth hormone terminal signal and so on. This modified vector was designated pEC(K) and supposed to effectively express short peptide. A well-defined single B-cell and T-cell epitope encoding gene of duck/hepatitis B virus has been synthesized as candidate epitope and cloned into pEC(K) plasmid, respectively. Transfection of the recombinant DNA into C(2)C(12) cell showed that modified plasmid could effectively express both the single B-cell and T-cell short epitope in the culture supernatant as confirmed by dot immunoblot assay (DIA). The recombinant single B and T-cell epitope-based DNA vaccine was administrated to C57BL/6 mice and could greatly induce specific humoral and CTL response. In addition, the specific antibody against B epitope could specifically bind to the DHBV particles. This report demonstrated that single epitope-based DNA vaccine using modified plasmid vector pEC(K) could induce effective specific immune responses and could be of great use for DNA vaccines.

CD8(+) T cells play a critical role in protective immunity against Hepatitis B Virus (HBV). Epitope-based DNA vaccines expressing HBV-dominant CTL epitopes can be used as candidate vaccines capable of inducing cytotoxic T Lymphocytes (CTL) responses. A plasmid DNA encoding a CTL epitope of HBV core antigen, HBc(18-27), was constructed. Intramuscular immunization of C57BL/6 mice with this DNA vaccine resulted in successful induction of HBV-specific CTL responses. In order to promote transportation of the peptide into endoplasmic reticulum (ER) to bind to MHC class I molecules for optimal class I antigen presentation, an ER targeting sequence (ERTS) was fused with the C(18-27) encoding gene. ERTS fusion significantly enhanced specific CD8(+) T cell responses in terms of CTL cytolysis as well as IFN-gamma secretion. This enhancement was correlated with promoted epitope presentation on target cell surface. We report here an enhanced immunogenicity of an epitope-based DNA vaccine using an ER targeting signal sequence, which has significant implications for future design of therapeutic HBV vaccine.

Inoculation with purified specific protein is usually the first step for preparation of monoclonal antibody (mAb). But it is quite difficult to obtain pure proteins especially with natural structures. Here we attempt to replace the protein inoculation with DNA immunization in the preparation of mAb. The eukaryotic expression vectors pcDNA3-PreS2/S and pVAX-PreS2/S encoding the HBV M protein were constructed and prepared for DNA immunization. Female BALB/c mice developed a well antibody response to the target antigen after muscle injection with corresponding plasmids. The mice with effective antibodies induced were used for preparation of mAb. We found the mice immunized with three administrations of pcDNA3-PreS2/S and boosted by intrasplenic injection with the same plasmid could be exploited for preparation of mAb. And positive hybridoma cell 2D3 that can secrete specific mAb was cloned and analyzed. Our studies demonstrate that gene immunization may provide a convenient and efficient way to prepare mAbs.

We report on the induction of primary and long-term memory cytotoxic T lymphocyte (CTL) responses against the nucleoprotein of the influenza virus A/PR8/34 in mice immunized with plasmid DNA targeted to B lymphocytes in the spleen. We found that the magnitude of the CTL response and the size of the pool of memory CTL was greater when the CTL response was induced in presence of T cell help. Interestingly, immunization with a signal sequence-competent transgene was markedly superior to immunization with a transgene lacking the endoplasmic reticulum (ER) targeting sequence, in inducing CTL. We also found a correlation between in vivo protection from lethal virus challenge and (1) the availability of T cell help and (2) ER targeting. Immunization of dendritic cell-deficient mice suggests that B lymphocytes function as antigen-presenting cells in this model of immunization. Collectively, the results suggest that somatic transgene immunization is a conceptually new approach to induce effective anti-viral CTL responses and to assess the parameters critical for long-lasting and protective CTL responses in vivo.