RESUMEN

To prepare a lipid nanoparticle (LNP)-based subunit vaccine of Mycobacterium tuberculosis (Mtb) antigen EsxV and study its immunological characteristics, the LNP containing EsxV and c-di-AMP (EsxV: C: L) was prepared by thin film dispersion method, and its encapsulation rate, LNP morphology, particle size, surface charge and polyphase dispersion index were measured. BALB/c mice were immunized with EsxV: C: L by nasal drops. The levels of serum and mucosal antibodies, transcription and secretion of cytokines in lung and spleen, and the proportion of T cell subsets were detected after immunization. EsxV: C: L LNPs were obtained with uniform size and they were spherical and negatively charged. Compared with EsxV: C immunization, EsxV: C: L mucosal inoculation induced increased sIgA level in respiratory tract mucosa. Levels of IL-2 secreted from spleen and ratios of memory T cells and tissue-resident T cells in mice were also elevated. In conclusion, EsxV: C: L could induce stronger mucosal immunity and memory T cell immune responses, which may provide better protection against Mtb infection.

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RESUMEN

To prepare a lipid nanoparticle (LNP)-based subunit vaccine of Mycobacterium tuberculosis (Mtb) antigen EsxV and study its immunological characteristics, the LNP containing EsxV and c-di-AMP (EsxV: C: L) was prepared by thin film dispersion method, and its encapsulation rate, LNP morphology, particle size, surface charge and polyphase dispersion index were measured. BALB/c mice were immunized with EsxV: C: L by nasal drops. The levels of serum and mucosal antibodies, transcription and secretion of cytokines in lung and spleen, and the proportion of T cell subsets were detected after immunization. EsxV: C: L LNPs were obtained with uniform size and they were spherical and negatively charged. Compared with EsxV: C immunization, EsxV: C: L mucosal inoculation induced increased sIgA level in respiratory tract mucosa. Levels of IL-2 secreted from spleen and ratios of memory T cells and tissue-resident T cells in mice were also elevated. In conclusion, EsxV: C: L could induce stronger mucosal immunity and memory T cell immune responses, which may provide better protection against Mtb infection.

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BACKGROUND: Subunit vaccines are appropriate vaccine candidates for the prevention of some infections. In this study, three immunogenic proteins of Mycobacterium tuberculosis, including HspX, Ppe44, and EsxV as a new construction, were expressed alone and as a fusion protein to develop a new vaccine candidate against tuberculosis infection. METHODS: To make the fusion protein, the three genes were linked together by AEAAAKEAAAKA linkers and inserted into pET21b and pET32b vectors. Escherichia coli (E. coli) Top10 cells were transformed with the plasmid, and the purified plasmid was used to transform E. coli BL21 cells. Protein expression was induced with IPTG. After optimizing protein expression, the recombinant proteins were purified by Ni-NTA chromatography. Protein purification was confirmed by SDS-PAGE and Western blotting with an anti-poly histidine-peroxidase monoclonal antibody against the 6His-tags at the proteins' C termini. RESULTS: Directional cloning was confirmed by polymerase chain reaction (PCR), restriction enzyme digestion, and sequencing. The highest expression of the tri-fusion protein and HspX were obtained by the addition of 0.2 mM of IPTG to E. coli BL-21 cells at 37 °C and 18 h of incubation. For Ppe44 and EsxV, the optimum expression conditions were 18 °C and 16 h of incubation. SDS-PAGE and Western blots confirmed that the desired proteins were produced. CONCLUSION: The three desired proteins and the fusion protein were successfully expressed and the conditions for optimum expression determined. These recombinant proteins will be evaluated as vaccine candidates against tuberculosis. Further studies are needed to evaluate the abilities of these proteins to induce strong immunological responses.

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Many materials such as aluminum hydroxide have been tried as adjuvants to compensate low inherent immunogenicity of subunit vaccines. The aim of this study was to evaluate the specific immune response following the administration of aluminum hydroxide nanoparticles with EsxV antigen. The physiochemical properties of the nanoparticle were characterized in vitro. After subcutaneous immunization, cytokine secretion patterns including IFN-gama,IL-4, and TGF-beta levels were measured by indirect enzyme linked immunosorbent assay (ELISA). Aluminum hydroxide-NPs were demonstrated excellent effects to raise of IFN-γ secretion in compare to EsxV alone. Administration of aluminum hydroxide nanoparticles stimulates strong cellular immune response and could be considered as appropriate adjuvant against TB infection.

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To identify factors contributing to the ability of tubercle bacilli to grow in the lung during active infection, we analyzed RNA expression patterns in bacteria present in patient sputum. Prominent among bacterial transcripts identified were those encoding secreted peptides of the Esat-6 subfamily that includes EsxK and EsxL (Rv1197 and Rv1198). H37Rv esxKL and esxJI transcripts were differentially expressed under different growth conditions, and disruption of these genes altered growth phase kinetics in typical laboratory batch broth cultures. These growth defects, including the reduced intracellular growth of an ΔesxKL mutant in primary human macrophages, were reversed by either low multiplicity co-infection or co-culture with wild-type bacteria, demonstrating the ability of the secreted factors to rescue isogenic mutants. Complementing either only esxL or esxI alone (Rv1198 or Rv1037c) also reduced observed growth defects, indicating these genes encode factors capable of contributing to growth. Our studies indicate that the Mycobacterium tuberculosis Mtb9.9 family secreted factors EsxL and EsxI can act in trans to modulate growth of intracellular bacteria, and are highly expressed during active human lung infection.