Formulation in DDA-MPLA-TDB Liposome Enhances the Immunogenicity and Protective Efficacy of a DNA Vaccine against Mycobacterium tuberculosis Infection

Abstract

Despite the vaccine Mycobacterium bovis Bacillus Calmette–Guérin is used worldwide, tuberculosis (TB) remains the first killer among infectious diseases. An effective vaccine is urgently required. DNA vaccine has shown prophylactic as well as therapeutic effects against TB, while its weak immunogenicity hinders the application. As a strong inducer of Th1-biased immune response, DMT, consisting of dimethyldioctadecylammonium (DDA) and two pattern recognition receptor agonists monophosphoryl lipid A and trehalose 6,6′-dibehenate (TDB), was a newly developed liposomal adjuvant. To elucidate the action mechanism of DMT and improve immunological effects induced by DNA vaccine, a new recombinant eukaryotic expression plasmid pCMFO that secretes the fusion of four multistage antigens (Rv2875, Rv3044, Rv2073c, and Rv0577) of Mycobacterium tuberculosis was constructed. pCMFO/DDA and pCMFO/DMT complexes were then prepared and their physicochemical properties were analyzed. The immunogenicity and protection against M. tuberculosis infection in vaccinated C57BL/6 mice were compared. Formulation of DNA and two agonists into the DDA liposome decreased zeta potential but increased the stability of storage, which resulted in a slower and longer-lasting release of DNA from the DNA–DMT complex than the DNA–DDA liposome. Besides Th1-biased responses, pCMFO/DMT vaccinated mice elicited more significantly CFMO-specific IL2+ TCM cell responses in the spleen and provided an enhanced and persistent protection against M. tuberculosis aerosol infection, compared to pCMFO/DDA and pCMFO groups. Therefore, the adjuvant DMT can release DNA and agonists slowly, which might attribute to the improved protection of DMT adjuvanted vaccines. pCMFO/DMT, a very promising TB vaccine, warrants for further preclinical and clinical trials.

Combinational PRR Agonists in Liposomal Adjuvant Enhances Immunogenicity and Protective Efficacy in a Tuberculosis Subunit Vaccine

“…Compared with the DDA liposome, an addition of TDB and/or MPLA into the DDA liposome did not result in the change of particle size and PDI ( Figure 1). In line with previous studies (25,26), the incorporation of MPLA into DDA vesicles resulted in a significant decrease of the surface charge, as demonstrated by the lower Zeta potential values of DM and DMT. The antigen CMFO, emulsified with different liposomes, resulted in a general trend of increased particle size and PDI while reduced zeta potential across all four formulations.…”

“…Different liposomes had a similar morphology and formed nearly spherical vesicles as our previous demonstrated by transmission electron microscopy (data not shown) (25). Compared with the DDA liposome, an addition of TDB and/or MPLA into the DDA liposome did not result in the change of particle size and PDI ( Figure 1).…”

“…Four liposomal formulations (Table S1), namely, DDA, DDA/ MPLA (DM), DDA/TDB (DT), and DMT, were prepared using the lipid film hydration method as previously described (25). Briefly, weighed amounts of DDA (Avanti Polar Lipids Inc., AL, USA), MPLA (Avanti), or TDB (Avanti) were first dissolved in chloroform/methanol (9:1 in volume).…”

Heterologous Boost Following Mycobacterium bovis BCG Reduces the Late Persistent, Rather Than the Early Stage of Intranasal Tuberculosis Challenge Infection

“…A recombinant replication-deficient adenovirus rAdCTT3H was packaged and constructed by co-transfecting plasmids pDC316-CTT3H and pBHGloxΔE1, 3 Cre into human embryonic kidney 293 (HEK293, ATCC® CRL-1573 ™) cells with Lipofactamine 2000 reagent (Invitrogen), as previously described ( 14 ). To construct DNA vaccine, the fusion gene was obtained by polymerase chain reaction (PCR) with the specific primers targeting to the plasmid pDC316-CTT3H and subcloned into the eukaryotic expression vector pVAX-1 (Thermo Fisher Scientific), as previously described ( 15 ). The resultant construction was named pCTT3H and the inserted gene was verified by enzyme digestion and DNA sequencing.…”

“…The purification and quantitation of recombinant adenovirus rAdCTT3H, recombinant protein CTT3H, and recombinant eukaryotic plasmid pCTT3H was performed as previously described, respectively ( 10 , 14 , 15 ). The concentration of the endotoxin in each purified products was detected by ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit (Genscript).…”