Clinical and immunological evaluation after BCG-id vaccine in leprosy patients in a 5-year follow-up study

Figures & data

Table 1 Clinical and laboratory data of leprosy patients before, during, and 5 years after MDT

Nº	ID	Age†	Sex	Race	BI-b	APGL-1-b‡,*	HP	MDT–MB/PB (doses)	RE	NE	BI-a	APGL-1-a‡,*
MB1	LCN	55	M	CAU	5+	13.5	LL	MB (24)	EN	–	1+	1.6
MB2	LSS	50	F	CAU	3+	1.8	BL	MB (12)	RR	Yes	Neg	0.3
MB3	JSA	33	M	CAU	4+	1.5	BL	MB (12)	–	–	Neg	0.1
MB4	CAL	39	F	CAU	4+	2.2	LL	MB (24)	–	–	Neg	0.5
MB5	AAR	38	M	CAU	4+	4.8	LL	MB (24)	EN	–	Neg	0.9
MB6	CAL	39	F	CAU	5+	5.5	LL	MB (12)	–	Yes	Neg	1.0
MB7	DCS	51	M	CAU	3+	2.6	LL	MB (24)	–	Yes	Neg	0.4
MB8	RI	69	M	CAU	3+	4.4	BL	MB (24)	RR	Yes	Neg	0.4
MB9	lARV	53	M	CAU	3+	6.5	BL	MB (12)	EN	–	Neg	0.5
MB10	APS	43	M	BLA	1+	1.1	BL	MB (4)	–	–	NP	NP
MB11	MLJ	45	F	BLA	3+	4.2	LL	MB (24)	EN	–	2+	4.1
PB1	RDM	43	M	CAU	Neg	0.6	TT	PB (6)	–	–	Neg	0.3
PB2	SLS	35	M	CAU	Neg	0.3	BT	PB (6)	–	Yes	Neg	0.3
PB3	JES	44	M	CAU	Neg	0	BT	PB (6)	–	–	Neg	0
PB4	CMO	61	F	CAU	Neg	0.3	TT	PB (6)	–	–	Neg	0.3
PB5	SR	51	M	CAU	Neg	0.7	TT	PB (6)	–	–	Neg	0.2
PB6	JPS	42	F	CAU	Neg	0.5	BT	MB (12)	RR	–	Neg	1.1
PB7	AFAS	21	M	CAU	Neg	0.9	BT	MB (12)	RR	–	Neg	0.8
PB8	MBSA	58	F	BLA	Neg	1.5	BT	PB (6)	–	–	Neg	0.8

Notes:

† Expressed as years;

‡ expressed as pg/mL;

* cut-off: 1.0/positive result: ≥1.0.

Abbreviations: MDT, multidrug therapy; Nº, number; ID, identification; BI-b, bacterial index before MDT; APGL-1-b, anti-PGL-1 before MDT; HP, histopathologic classification; MB, multibacillary; PB, paucibacillary; RE, reactional episodes; NE, neuritis; BI-a, bacterial index after MDT; APGL-1-a, anti-PGL-1 after MDT; M, male; F, female; CAU, caucasian; BLA, black; Neg, negative; LL, lepromatous; BL, borderline-lepromatous; TT, tuberculoid; BT, borderline-tuberculoid; EN, erythema nodosum; RR, reverse reaction; NP, not performed.

Table 2 Quantification of IL-10, IFN-γ, TNF-α, and IL-6 (pg/mL) in PBMC supernatants from leprosy MB patients before and 30 days after BCG-id

Patients	Nonadherent cells – Con-A (50 μg/mL)				Adherent cells – LPS (5 mg/mL)
	Before BCG-id		After BCG-id		Before BCG-id		After BCG-id
	IL-10	IFN-γ	IL-10	IFN-γ	TNF-α	IL-6	TNF-α	IL-6
MB1	1089.3	257.3	1130	6964.4	NP	NP	NP	NP
MB2	402.7	74,395	256	71,602.8	NP	NP	NP	NP
MB3	746	89,475.6	1746	99,475.2	747.2	2347.5	1756.9	3341.7
MB4	2778	12,207	2430.1	788.4	NP	NP	NP	NP
MB5	1402.1	35,228.6	1471.4	696.3	3277.7	3554.1	6337.6	6669.3
MB6	1005.4	903.7	2005.4	1902.5	2151.6	20,262.3	4151.3	30,262.3
MB7	517.1	27,358.9	1038.5	2329.5	175	498.9	331.9	2739.9
MB8	ND	27,005.9	ND	37,015.6	697.3	1536	1696.5	2536
MB9	706.5	683.1	1443.7	592	264.7	1127	245.6	1968.2
MB10	1592.6	3586	728.5	4441.1	420.4	4727.1	181.7	930.2
MB11	2181	819.7	1148.4	5959.4	433.9	2990.7	1566.2	10,407.3
Median	1047.3	7896.5	1296.0	3385.3	565.6	2669.1	1631.7	3043.7
P-value			0.339	0.789			0.017	0.056

Abbreviations: IL, interleukin; IFN-γ, interferon-gamma; TNF-α, tumor necrosis factor-alpha; PBMC, peripheral blood mononuclear cells; MB, multibacillary; BCG-id, bacillus Calmette–Guérin – intradermal; Con-A, Concanavalin-A; LPS, lipopolysaccharides; ND, not detected; NP, not performed.

Figure 1 Graphs representing the median levels of IL-10, IFN-γ, TNF-α and IL-6 (pg/mL) in PBMC supernatants from leprosy patients before and after BCG-id.

Abbreviations: IL, interleukin; IFN-γ, interferon-gamma; TNF-α, tumor necrosis factor-alpha; PBMC, peripheral blood mononuclear cells; BCG-id, bacillus Calmette– Guérin – intradermal; MB, multibacillary; Con-A, Concanavalin-A; PB, paucibacillary; LPS, lipopolysaccharides.

Table 3 Quantification of IL-10, IFN-γ, TNF-α, and IL-6 (pg/mL) in PBMC supernatants from leprosy PB patients before and 30 days after BCG-id

Patients	Nonadherent cells – Con-A (50 μg/mL)				Adherent cells – LPS (5 mg/mL)
	Before BCG-id		After BCG-id		Before BCG-id		After BCG-id
	IL-10	IFN-γ	IL-10	IFN-γ	TNF-α	IL-6	TNF-α	IL-6
PB1	1005.4	16,355.1	2099	15,790.5	NP	NP	NP	NP
PB2	1709.9	14,119.7	2259.2	8542.2	NP	NP	NP	NP
PB3	877.4	37,348.7	1871.3	47,347.5	1499.5	7837.3	2450.6	17,836.5
PB4	1750.5	15,617.6	169.4	29,082.6	302.5	609.9	125.7	150.7
PB5	1524.2	27,566.3	213.4	29,344.3	1102.8	6629.5	833.9	4765.7
PB6	2561.8	11,469.6	1880.2	26,851.9	154.9	342.8	418.2	1049.8
PB7	987.6	1910.7	1978.5	2911.9	1199.6	4823.6	2198.4	14,822.4
PB8	2176.1	16,761.9	2012.5	42,502.7	319.6	1837	2844.1	13,950
Median	1617.0	15,986.3	1933.9	27,967.2	711.2	3330.3	1516.7	9357.8
P-value			0.513	0.037			0.076	0.051

Abbreviations: IL, interleukin; IFN-γ, interferon-gamma; TNF-α, tumor necrosis factor-alpha; PBMC, peripheral blood mononuclear cells; PB, paucibacillary; BCG-id, bacillus Calmette–Guérin – intradermal; Con-A, Concanavalin-A; LPS, lipopolysaccharides; ND, not detected; NP, not performed.

Table 4 Quantification of IL-17 (pg/mL) in PBMC supernatants from leprosy patients before and 30 days after BCG-id

Patients	Before BCG-id	After BCG-id
Nonadherent cells – BCG (20 μg/mL)
MB1	7.0	10.5
MB2	1.4	2.9
MB3	14.1	NP
MB4	5.4	10.0
MB5	4.4	62.6
MB6	3.9	NP
MB7	4.9	NP
PB1	7.0	36.9
PB2	3.4	24.6
PB3	0.4	NP
PB4	14.1	15.7
PB5	8.1	42.5
PB6	36.4	70.8
Median MB	4.6	21.6
Median PB	11.6	38.7
P-value MB		0.153
P-value PB		0.008

Abbreviations: IL, interleukin; PBMC, peripheral blood mononuclear cells; BCG, bacillus Calmette–Guérin intradermal; MB, multibacillary; PB, paucibacillary; NP, not performed.

Figure 2 Photomicrograph of a skin biopsy. (A) Infiltrate of histiocytes surrounded by lymphocytes in the dermis in a case of MB leprosy before the BCG-id (HE, ×100). (B) Tendency towards granuloma formation in a case of MB leprosy after the BCG-id (HE, ×100). (C) Immunoreactivity for INFγ in a case of MB leprosy before the BCG-id (NovoLink staining with hematoxylin counterstaining, ×50). (D) Immunoreactivity for INFγ in a case of MB leprosy after the BCG-id (NovoLink staining with hematoxylin counterstaining, ×50). (E) Immunoreactivity for IL-1 in a case of MB leprosy before the BCG-id (NovoLink staining with hematoxylin counterstaining, ×100). (F) Immunoreactivity for IL-1 in a case of MB leprosy after the BCG-id (NovoLink staining with hematoxylin counterstaining, ×100). (G) Immunoreactivity for IL-10 in a case of MB leprosy before the BCG-id (NovoLink staining with hematoxylin counterstaining, ×100). (H) Immunoreactivity for IL-10 in a case of MB leprosy after the BCG-id (NovoLink staining with hematoxylin counterstaining, ×100).

Abbreviations: MB, multibacillary; BCG-id, bacillus Calmette–Guérin – intradermal; HE, hematoxilin eosin; INF-γ, interferon-gamma; IL, interleukin.

Figure 3 Photomicrograph of a skin biopsy. (A) Immunoreactivity for IL-12 in a case of MB leprosy before the BCG-id (NovoLink staining with hematoxylin counterstaining, ×100). (B) Immunoreactivity for IL-12 in a case of MB leprosy after the BCG-id (NovoLink staining with hematoxylin counterstaining, ×100). (C) Immunoreactivity for TGF-β in a case of MB leprosy before the BCG-id (NovoLink staining with hematoxylin counterstaining, ×100). (D) Immunoreactivity for TGF-β in a case of MB leprosy after the BCG-id (NovoLink staining with hematoxylin counterstaining, ×100). (E) Immunoreactivity for TNF-α in a case of MB leprosy before the BCG-id (NovoLink staining with hematoxylin counterstaining, ×100). (F) Immunoreactivity for TNF-α in a case of MB leprosy after the BCG-id (NovoLink staining with hematoxylin counterstaining, ×100). (G) Immunoreactivity for TNF-α in a case of PB leprosy before the BCG-id (NovoLink staining with hematoxylin counterstaining, ×100). (H) Immunoreactivity for TNF-α in a case of PB leprosy after the BCG-id (NovoLink staining with hematoxylin counterstaining, ×100).

Abbreviations: IL, interleukin; MB, multibacillary; BCG-id, bacillus Calmette–Guérin – intradermal; TGF-β, transforming growth factor beta; TNF-α, tumor necrosis factor-alpha.