DNA Vaccine Constructs Expressing Mycobacterium tuberculosis-Specific Genes Induce Immune Responses

Abstract

RD1 PE35, PPE68, EsxA, EsxB and RD9 EsxV genes are present in Mycobacterium tuberculosis genome but deleted in Mycobacterium bovis BCG. The aim of this study was to clone these genes into DNA vaccine vectors capable of expressing them in eukaryotic cells as fusion proteins, fused with immunostimulatory signal peptides of human interleukin‐2 (hIL‐2) and tissue plasminogen activator (tPA), and evaluate the recombinant DNA vaccine constructs for induction of antigen‐specific cellular immune responses in mice. DNA corresponding to the aforementioned RD1 and RD9 genes was cloned into DNA vaccine plasmid vectors pUMVC6 and pUMVC7 (with hIL‐2 and tPA signal peptides, respectively), and a total of 10 recombinant DNA vaccine constructs were obtained. BALB/c mice were immunized with the parent and recombinant plasmids and their spleen cells were tested for antigen‐induced proliferation with antigens of M. tuberculosis and pure proteins corresponding to the cloned genes. The results showed that antigen‐specific proliferation responses were observed for a given antigen only with spleen cells of mice immunized with the homologous recombinant DNA vaccine construct. The mice immunized with the parent plasmids did not show positive immune responses to any of the antigens of the cloned genes. The ability of the DNA vaccine constructs to elicit cellular immune responses makes them an attractive weapon as a safer vaccine candidate for preventive and therapeutic applications against tuberculosis.

Molecular Cloning, Expression, Purification and Immunological Characterization of Proteins Encoded by Regions of Difference Genes of Mycobacterium tuberculosis

“…Groups of 6-8 week old BALB/c mice (five mice in each group) were immunized intramuscularly with three doses of purified parent or recombinant plasmid DNA, each dose containing 100 μg of DNA, and given 3 weeks apart. After 3 weeks of the third immunization, spleen cells were isolated from each immunized mouse for cellular immune responses using antigen-induced proliferation as an indicator (Hanif et al, 2010c). The results demonstrated that spleen cells of mice un-immunized or immunized with the parent plasmids did not show positive antigen-induced proliferation responses to any of the antigens corresponding to the cloned genes (Hanif et al, 2010c).…”

“…2, 3). In this way, a total of 10 recombinant DNA plasmids (five for each vector) were constructed (Hanif et al, 2010c). To study the expression and immunogenicity of the RD1 and RD9 proteins cloned in the recombinant vaccine constructs of pUMVC6 and pUMVC7, immunization studies were performed in mice.…”

“…pUMVC6 and pUMVC7, to clone and express M. tuberculosis-specific RD proteins and studied antigen-specific cellular immune responses after immunization of mice with the recombinant plasmids (Hanif et al, 2010c). The plasmid vectors pUMVC6 and pUMVC7 are eukaryotic expression vectors, which have been prepared by University of Michigan Vector Core (UMVC) and made commercially available by Aldevron, USA.…”

Construction and expression of a recombinant eukaryotic expression plasmid containing the preS1-preS2-S genes of hepatitis B virus and the granulocyte-macrophage colony stimulating factor gene: A study of its immunomodulatory effects

“…It is relatively simple to clone and is a purified DNA with no need for a synthetic protein in vitro, and can sustain long-term immune efficacy (5). The recombinant DNA vaccine is also relatively cost-effective and convenient to transport and preserve, thus it is a promising approach for the vaccine development.…”

Enhanced immune response of a bicistronic DNA vaccine expressing fusion antigen Hsp65-Esat-6 of Mycobacterium Tuberculosis with GM-CSF as a molecular adjuvant

“…Currently, DNA vaccine has gained more attention with greater development since it maintains a long immune response and is safe as a recombinant subunit vaccine. It is effective as a live attenuated vaccine in systemic immune response, with easier cloning of DNA molecule and no requirement of protein synthesis's in vitro, extraction and purification (Hanif et al 2010). The MTB hot shock protein 65 (heat shock protein 65, Hsp65), one of earliest MTB antigen studied, may have the intense protection immune response in the animal in vivo to MTB's infection (Yoshida et al 2006;Sechi et al 2006;Okada et al 2007).…”