This study evaluated the immune response elicited by a ubiquitin (Ub)-fused MPT64 DNA vaccine against Mycobacterium tuberculosis. BALB/c mice were vaccinated with plasmid DNA encoding MPT64 protein, Ub-fused MPT64 DNA vaccine (UbGR-MPT64), and negative DNA vaccines, respectively. MPT64 DNA vaccine immunization induced a Thl-polarized immune response. The production of Thl-type cytokine (interferon-gamma [IFN-γ]) and proliferative T cell responses were enhanced significantly in mice immunized with UbGR-MPT64 fusion DNA vaccine, compared with nonfusion DNA vaccine. Moreover, this fusion DNA vaccine also resulted in an increased relative ratio of IgG2a to IgGl and the cytotoxicity of T cells. IFN-γ intracellular staining of splenocytes indicated that UbGR-mpt64 fusion DNA vaccine activated CD4+ and CD8+ T cells, particularly CD8+ T cells. Thus, this study demonstrated that the UbGR-MPT64 fusion DNA vaccine inoculation could improve antigen-specific cellular immune responses, which is helpful for protection against TB.
"The intracellular localization of an antigen can influence the magnitude and quality of humoral immune response and can also target the response to CD8+ or CD4+ T cells. In this regard, antigen targeting to different cellular processing compartments may improve its presentation by MHC I or MHC II molecules and enhance specific immune response –. In addition, the secretion of cellular proteins was reported to modulate the immunological responses. "
[Show abstract][Hide abstract] ABSTRACT: We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field.
PLoS ONE 06/2014; 9(6):e99887. DOI:10.1371/journal.pone.0099887 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We analyzed different vaccine approaches aimed at enhancing CD4(+)- and CD8(+)-dependent responses against hepatitis C virus (HCV) core antigen. Specific DNA vectors expressing various forms of the core in fusion with the ubiquitin or the lysosome-associated membrane protein (LAMP) were generated. These expressed the full-length wildtype core; the full-length core expressed as a covalent fusion with the ubiquitin; the full-length core expressed as a noncovalent fusion with the ubiquitin and containing a N-stabilizing or N-destabilizing residue; and the full-length core expressed as a fusion with the LAMP sequence. In vitro expression levels of the different plasmids differed by as much as tenfold. After injection into mice, none of the plasmids yielded a detectable antibody response, whereas core-specific cytotoxic T-lymphocyte (CTL) activity could be observed with all plasmids as long as 21 weeks postimmunization. No increase in CTL activity (ranging from 7% to 34% specific lysis) was observed with the ubiquitin-fusion-expressed core antigens compared with the wildtype core. The lowest CTL activity (< 5% specific lysis) was observed with the LAMP fusion. This vector was nonetheless unable to induce a detectable proliferative response. Screening of 10 different putative CTL peptide epitopes failed to reveal newly targeted epitopes when the core-fusion plasmids were used compared with the wildtype core-expressing plasmid. These data underline the difficulty in optimizing anti-core cellular immune response using molecular targeting strategies in DNA-based vaccination.
DNA and Cell Biology 08/1999; 18(8):611-21. DOI:10.1089/104454999315024 · 2.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Heterologous prime-boost regimens utilizing BCG as a prime vaccine probably represent the best hope for the development of novel tuberculosis (TB) vaccines. In this study, we examined the immunogenicity and protective efficacy of DNA vaccine (pcD685A) expressing the fusion protein of Ag85A and ESAT-6 (r685A) and its booster effects in BCG-immunized mice. The recombinant r685A fusion protein stimulated higher level of antigen-specific IFN-γ release in tuberculin skin test- (TST-) positive healthy household contacts of active pulmonary TB patients than that in TST-negative population. Vaccination of C57BL/6 mice with pcD685A resulted in significant protection against challenge with virulent Mycobacterium tuberculosis H37Rv when compared with the control group. Most importantly, pcD685A could act as a BCG booster and amplify Th1-type cell-mediated immunity in the lung of BCG-vaccinated mice as shown the increased expression of IFN-γ. The most significant reduction in bacterial load of both spleen and lung was obtained in mice vaccinated with BCG prime and pcD685A DNA booster when compared with BCG or pcD685A alone. Thus, our study indicates that pcD685A may be an efficient booster vaccine against TB with a strong ability to enhance prior BCG immunity.
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