Low cost tuberculosis vaccine antigens in capsules: expression in chloroplasts, bio-encapsulation, stability and functional evaluation in vitro

Abstract

Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading fatal infectious diseases. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, three candidate antigens, ESAT-6 (6 kDa early secretory antigenic target) and Mtb72F (a fusion polyprotein from two TB antigens, Mtb32 and Mtb39) fused with cholera toxin B-subunit (CTB) and LipY (a cell wall protein) were expressed in tobacco and/or lettuce chloroplasts to facilitate bioencapsulation/oral delivery. Site-specific transgene integration into the chloroplast genome was confirmed by Southern blot analysis. In transplastomic leaves, CTB fusion proteins existed in soluble monomeric or multimeric forms of expected sizes and their expression levels varied depending upon the developmental stage and time of leaf harvest, with the highest-level of accumulation in mature leaves harvested at 6PM. The CTB-ESAT6 and CTB-Mtb72F expression levels reached up to 7.5% and 1.2% of total soluble protein respectively in mature tobacco leaves. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Western blot analysis of lyophilized lettuce leaves stored at room temperature for up to six months showed that the CTB-ESAT6 fusion protein was stable and preserved proper folding, disulfide bonds and assembly into pentamers for prolonged periods. Also, antigen concentration per gram of leaf tissue was increased 22 fold after lyophilization. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of the ESAT-6 antigen. GM1-binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to bind with the GM1-ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB booster vaccine with potential for long-term storage at room temperature. To our knowledge, this is the first report of expression of TB vaccine antigens in chloroplasts.

Figure 1. Schematic representation of chloroplast transformation vectors harboring tuberculosis candidate vaccine antigens.

Ls, Lactuca sativa; Nt, Nicotiana tabacum; pLD- vector components: the homologous Nicotiana tabacum chloroplast genome flanking sequence comprising of 16S 3′ end sequences and complete trnI, trnA genes; pLs- vector components: 16S trnI, the homologous Lactuca sativa chloroplast genome long flanking sequence containing 16S 3′end sequences and full length trnI gene; trnA 23S, trnA gene and 5′end of the 23S ribosomal RNA subunit; Prrn, ribosomal RNA operon promoter with GGAGG ribosome binding site; aadA, aminoglycoside 3′-adenylytransferase gene; TrbcL, 3′ untranslated region (Utr) of rbcL gene; PpsbA, promoter and 5′ Utr of psbA gene; TpsbA, 3′ Utr of psbA gene; CTB-ESAT6, coding sequence of cholera toxin B subunit fused to ESAT6 (6 kDa early secretory antigenic target); CTB-Mtb72F, coding sequence of cholera toxin B subunit fused to a recombinant fusion polyprotein from two TB antigens Mtb32 and Mtb39; LipY, coding sequence of a TB cell wall protein with lipase activity.

Figure 2. Southern blot analysis to evaluate site-specific transgene integration and homoplasmy in transplastomic plants.

An autoradiograph of Southern blot hybridized with flanking sequence probe comprising of trnI and trnA gene sequences. A, Homoplasmy in CTB-ESAT6 tobacco (Nt) transplastomic lines. B, Heteroplasmy in CTB-Mtb72F tobacco (Nt) transplastomic lines. C, Homoplasmy in CTB-ESAT6 lettuce (Ls) transplastomic lines. D, Homoplasmy in LipY tobacco (Nt) transplastomic lines. Lane N, untransformed plant; Lane 1 to 6, transplastomic lines; Lane M, 1 Kb+ DNA ladder.

Figure 3. Western blot analysis to detect expression of recombinant proteins in transplastomic plants.

Immunoblots with anti CTB antibody showing presence of expected size fusion protein band in CTB-ESAT6 tobacco (A) and CTB-ESAT6 lettuce (B) transplastomic lines. Immunoblots showing pentameric CTB-ESAT6 fusion protein detection with protein extracted under non-reducing/non-denaturing conditions and loaded on SDS-PAGE without boiling (C). Immunoblots showing distribution of recombinant fusion protein in the homogenate, supernatant and pellet fractions of leaf extract from CTB-ESAT6 tobacco (D), CTB-ESAT6 lettuce (E) and CTB-Mtb72F tobacco (F) transplastomic plants. (N, Untransformed; H, homogenate; S, S1 & S2: supernatant; P, pellet; 1 to 4, transplastomic plants; C, purified bacterial CTB standard; C1, C2, C3 & C4: CTB standards 25, 50, 75, 100 ng for densitometry). Immunoblot using rat anti-LipY antibody showing presence of expected band in LipY transplastomic tobacco plants (G). Lane1, untransformed; Lanes 2 to 7, transformants. Immunoblot using rat anti-LipY antibody showing presence of expected band in LipY transplastomic lettuce plants (H). Lane 1, Supernatant fraction of leaf extract from transplastomic plant; Lane 2, Homogenate fraction of leaf extract from transplastomic plant; Lane 3, untransformed.

Figure 4. Quantitation of vaccine antigens in transplastomic lines at different developmental stages and harvesting time.

A, Expression levels of tobacco CTB-ESAT6 (Nt) protein in percent total soluble protein (TSP) as a function of leaf age and harvesting time under normal growth conditions determined by ELISA. B, Expression levels of tobacco CTB-Mtb72F (Nt) protein in percent TSP in young, mature and old leaves collected from the transplastomic lines by ELISA. Expression levels of CTB-ESAT6 (Ls) protein in lettuce transplastomic lines in percent total protein (TP) by densitometry at different leaf developmental stages (C) and at various time of harvest (D). 1 to 3, transplastomic lines. Expression levels of CTB-ESAT6 (Nt) and CTB-Mtb72F (Nt) are reported as percentage of the total soluble protein whereas CTB-ESAT6 (Ls) is in percentage of the total protein. Initially, ELISA was performed to quantify CTB-ESAT6 (Nt) in the total soluble protein but subsequent analysis of pellet, supernatant and homogenate fractions of leaf extracts detected fusion protein in the insoluble fraction. Therefore, CTB-ESAT6 (Ls) fusion protein was estimated based on percentage of total leaf protein. The CTB-Mtb72F fusion protein was quantitated based on percentage of the total soluble protein as the fusion protein existed only in the soluble fraction. Data presented is mean ± SD of triplicate experiments. Protein was extracted from three independent plants and amount of CTB-fusion protein was quantified either by ELISA or by densitometry on western blots. Three independent measurements were made to quantify expression levels of fusion protein and a mean ± SD value was plotted on the graph.

Figure 5. Lyophilization of CTB-ESAT6 lettuce leaves for long term storage and enrichment of antigenic fusion protein.

A, Preparation of capsules containing lyophilized powdered lettuce leaves expressing CTB-ESAT6 fusion protein for use as oral vaccine. B, Immunoblot comparing amount of CTB-ESAT6 fusion protein in fresh and lyophilized lettuce leaves. Equal amount of lyophilized and fresh leaves was used for protein extraction and equal volume of protein extract was loaded with dilutions of 1x, 10x and 20x; CTB, 50 ng CTB standard. C, CTB-ESAT6 expression levels in µg/g of fresh and lyophilized leaves. D, CTB-ESAT6 protein concentration (µg/g) in lyophilized leaves stored 0–6 months at room temperature. Error bars represent standard deviation of mean.

Figure 6. Functional evaluation of CTB-ESAT6 fusion protein in fresh and lyophilized lettuce leaves.

A, Ganglioside GM1 ELISA binding assay of CTB-ESAT6 derived from total leaf protein (TLP) of transplastomic plants. 1, 2∶15 µg TLP from CTB-ESAT6 transplastomic plants; WT, 15 µg TLP from untransformed lettuce plant; CTB, purified CTB standard; BSA, 50 ng bovine serum albumin. B, Ganglioside GM1 ELISA binding assay of CTB-ESAT6 from fresh and lyophilized lettuce leaves. Fresh and lyophilized CTB-ESAT6 lettuce leaf extracts starting from 15 µg equal concentrations and with decreasing two fold dilutions were analyzed for binding assay; Wild type, untransformed leaf extract; Purified CTB, 10 ng purified CTB standard.

Figure 7. Hemolysis assay with antibody affinity purified CTB-ESAT6.

A, Immunoblot with CTB antibody to confirm the presence of CTB-ESAT6 fusion protein. S1, S2, S3∶12.5, 25, 37.5 ng CTB standard; E1, E2, E3∶5 µl of different elution fractions; B4IP, 10 µg of total protein extract from CTB-ESAT6 transplastomic line before purification; WT, 10 µg of total protein extract from untransformed lettuce plant. B, Silver stained gel of affinity purified CTB-ESAT6 fusion protein. Elution1, 1st elution; Elution2, 2nd elution; E1, E2, E3, 2E1∶20 µl of different elution fractions of CTB-ESAT6; M, 0.4 µl protein standard marker. C, Hemolysis assay of ESAT6 with purified fusion protein CTB-ESAT6. Water causes complete hemolysis; E1 w/o sol, 40 µg/ml partially purified CTB-ESAT6 protein after 1st elution without solubilization; E1, 40 µg/ml partially purified CTB-ESAT6 protein after 1st elution with solubilization; 2E1, 20 µg/ml partially purified CTB-ESAT6 protein after 2nd elution with solubilization; PBS, phosphate buffer saline control; Elution/sol buffer, elution buffer used for protein elution/solubilisation; CTB, 40 µg/ml purified CTB. Standard deviations are represented by error bars of mean. Values are of 2 independent experiments in duplicates.