Identification and evaluation of the novel immunodominant antigen Rv2351c from Mycobacterium tuberculosis

Figures & data

Table 1 T-cell epitopes predicted by TEpredict and IEDB

Peptide ID	Location (start–end)	Amino-acid sequence	Immunogenicity score	Number of bound HLA alleles
1	6–14	FLTKLTGAG	−0.1272	2
2	18–26	FLMDWAAPV	0.24439	8
3	46–54	IVLLMQENR	−0.21408	4
4	76–84	FQQMGWNPM	0.01527	6
5	134–142	WLPAQATTR	0.01524	2
6	146–152	YVPLTMGYY	−0.11582	4
7	163–171	YLLADTFTI	0.21539	7
8	169–177	FTICDGYHC	0.05823	4
9	180–188	LTGTLPNRL	0.0536	2
10	236–244	YQNKGLGRF	−0.1377	4
11	281–289	FAADVRANR	0.15318	4
12	295–303	WLVPNILQS	0.04418	2
13	315–323	VSMVTALRI	0.08005	3
14	317–325	MVTALRILL	0.19573	4
15	323–331	ILLSNPAVW	−0.12967	3
16	363–371	FVTVPNIDA	0.16253	2
17	390–398	CIVISPYSR	−0.124	3
18	434–442	VVGDMTSAF	−0.2179	6
19	473–481	VVLGTTDGA	0.14336	2
20	485–493	IPYRVPYPQ	0.07104	6

Figure 1 SDS-PAGE and Western blot analysis to detect the purified recombinant Rv2351c protein expression. Lanes: A/E, Standard protein marker; B, non-induced pET-32a-Rv2351c; C, induced pET-32a-Rv2351c; and D/F, purified recombinant Rv2351c protein.

Table 2 Baesline data of the participants enrolled in the study

	PTB	Non-TB	HD	Total
Total gender	61	38	60	159
Male	43	22	26	91
Female	18	16	34	68
Age, years
Median (±SD)	43.5±18.0	54.3±19.3	24.7±2.0^a,^b

Abbreviations: pulmonary tuberculosis, PTB; patients with pulmonary disease but not TB, Non-TB; healthy donors, HD; standard deviation, SD.

^aP<0.05 (median age between healthy donors and PTB group).

^bP<0.05 (median age between healthy donors and PTB group).

Figure 2 Response magnitude of different subjects against cocktail peptides and Rv2351c in the ELISpot assay. Sixty-one patients with active TB, 38 patients with no TB and 55 healthy donors were enrolled to evaluate the T-cell response to cocktail peptides (Rv3615c, ESAT-6 and CFP-10) and the Rv2351c protein. Responses against the cocktail peptides and the Rv2351c protein were obtained through the T-SPOT.TB assay. The dots represent the response in each case under stimulation with cocktail peptides and Rv2351c. The thick line represents the average response of each group. P was calculated by t-test to evaluate the statistically significant differences (*P<0.05; **P<0.001).

Table 3 Comparison of results using the bacteriological test results as ‘gold standard’ for TB diagnosis

	T-SPOT.TB	ELISpot-2351c	P-value
Sensitivity	98.2% (56/57)	61.4% (35/57)	0.000
Specificity	94.6% (88/93)	91.4% (85/93)	0.388
Youden index	0.928	0.528
Kappa value	0.916	0.554

Note: 57 TB patients were diagnosed positive and 93 were diagnosed negative with the ‘gold standard’.

Figure 3 Antibody response against Ag85B and Rv2351c in BALB/c mice immunized with Ag85B or Rv2351c conjugate in DDA/poly (I:C) adjuvant. Serum samples were analyzed for the presence of anti-Ag85B and anti-Rv2351c antibodies via ELISA. The isotype profile of the antibodies was characterized using conjugated secondary antibodies specific for IgG, IgG1 and IgG2a. The data are plotted as geometric mean±SD log₁₀ end point titer. P was calculated by t-test to evaluate the statistically significant differences (*P<0.05; **P<0.001).

Figure 4 Evaluation of cytokine secretion by splenocytes from immunized mice, isolated and co-cultured with Ag85B or Rv2351c. The splenocytes were prepared four weeks after the mice were immunized with Ag85B or Rv2351c (three times, 2-week intervals). The splenocytes (1 × 10⁶) were then co-cultured with Ag85B (5 μg/mL) or Rv2351c (5 μg/mL) for 72 h before the levels of the cytokines (A) IFN-γ, (B) IL-2 and (C) IL-4 were measured in the culture supernatants using commercial ELISA kits. The data for cytokine secretion are presented as the mean±SD of two independent experiments. The level of statistical significance for differences between the negative control groups and the Ag85B or Rv2351c groups was determined using the t-test (*P<0.05; **P<0.001).