HCV-specific immune responses induced by CIGB-230 in combination with IFN-α plus ribavirin

Table 1 Demographic, virological and histological data of patients included in the trial n (%)

		Control group	Group E6	Group E9	Group L6	Group L9
Demographics1 and virological response
		n = 30	n = 16	n = 16	n = 15	n = 15
Age (yr)	mean ± SD	48.5 ± 7.4	47.4 ± 9.1	42.6 ± 7.9	47.3 ± 7.5	45.3 ± 7.3
Sex	Feminine	12 (40)	11 (68.8)	8 (50.0)	11 (73.3)	9 (60)
	Masculine	18 (60)	5 (31.3)	8 (50.0)	4 (26.7)	6 (40)
Body Mass Index	mean ± SD	26.7 ± 2.8	24.8 ± 3.5	28.6 ± 4.6	25.3 ± 5.0	24.9 ± 4.4
Race	White	16 (53.3)	12 (75.0)	11 (68.8)	10 (66.7)	8 (53.3)
	Black	4 (13.3)	3 (18.8)	3 (18.8)	4 (26.7)	3 (20.0)
	Mixed	10 (33.3)	1 (6.3)	2 (12.5)	1 (6.7)	4 (26.7)
Response to treatment2	cEVR	18 (60.0)	10 (62.5)	9 (56.3)	7 (46.7)	4 (26.7)
	SVR	14 (46.7)	8 (50)	8 (50)	7 (46.7)	2 (13.3)
Histology3
Week 0	n	30	16	16	15	15
Necroinflammation	mean ± SD	6.7 ± 3.6	6.4 ± 3.2	6.1 ± 3.3	5.1 ± 3.4	7.0 ± 4.1
Fibrosis		1.6 ± 1.4	1.9 ± 1.3	1.4 ± 1.4	1.6 ± 1.3	2.3 ± 1.8
Week 72	n	19	13	14	9	8
Necroinflammation	mean ± SD	3.3 ± 2.5	3.8 ± 2.2	3.2 ± 2.8	4.9 ± 3.0	5.6 ± 3.2
Fibrosis		1.3 ± 1.5	2.1 ± 1.3	1.1 ± 1.3	2.1 ± 1.6	2.5 ± 1.6

¹Corresponding to baseline;

²By intention to treat;

³According to Ishak score[17]. cEVR: Complete early viral responders; SVR: Sustained viral responders.

Table 2 Humoral and neutralizing antibody response

Antigen			Control group			E6 group			E9 group			L6 group			L9 group
			Week 0	Week 48	P value	Week 0	Week 48	P value	Week 0	Week 48	P value	Week 0	Week 48	P value	Week 0	Week 48	P value
Humoral response
Capsid	IgM	Seroc	96.1	96.1	-	100.0	100.0	-	100.0	100.0	-	100.0	100.0	-	100.0	100.0	-
		Titer	108.9 ± 96.4	57.3 ± 81.9	0.002	269.3 ± 313.5	107.4 ± 105.3	0.003	210.8 ± 223.0	98.3 ± 104.2	0.008	310.5 ± 338.4	273.8 ± 530.7	ns	174.9 ± 182.5	170.7 ± 221.6	ns
	IgA	Seroc	84.6	73.1	ns	86.6	66.6	ns	86.6	86.6	-	92.8	85.7	ns	100.0	91.6	ns
		Titer	12.0 ± 20.8	10.0 ± 16.2	0.020	48.2 ± 76.9	23.6 ± 26.6	0.010	38.3 ± 49.2	29.3 ± 34.4	ns	34.1 ± 38.5	24.8 ± 41.3	0.006	30.3 ± 25.6	22.9 ± 26.9	ns
	IgG	Seroc	96.1	96.1	-	100.0	100.0	-	100.0	100.0	-	100.0	100.0	-	91.6	91.6	-
		Titer	30056.0 ± 39875.0	17913.7 ± 24413.0	< 0.001	49443.7 ± 40431.0	24135.7 ± 23793.0	0.002	30604.8 ± 22036.0	28932.5 ± 24813.0	ns	42400.4 ± 73634.0	31502.8 ± 62453.0	0.040	52700.6 ± 69336.1	33379.0 ± 52006.1	0.010
E2	IgM	Seroc	34.6	19.2	ns	33.3	26.6	ns	13.3	13.3	ns	57.1	35.7	ns	25.0	33.3	ns
		Titer	7.1 ± 3.5	9.7 ± 14.5	ns	7.8 ± 4.7	7.8 ± 2.4	ns	5.9 ± 2.0	5.9 ± 1.9	ns	8.7 ± 3.7	8.8 ± 5.5	ns	11.4 ± 19.1	9.4 ± 7.2	ns
	IgA	Seroc	15.3	11.5	ns	6.6	6.6	ns	6.6	0.0	-	14.2	0.0	-	0.0	0.0	-
		Titer	5.8 ± 1.8	5.6 ± 1.6	ns	5.3 ± 1.3	5.3 ± 1.3	ns	5.3 ± 1.3	5.00 ± 0	ns	5.7 ± 1.8	5.0 ± 0	ns	5.0 ± 0.0	5.0 ± 0.0	ns
	IgG	Seroc	61.5	80.7	ns	46.4	66.6	ns	86.6	86.6	ns	71.4	71.4	ns	66.6	83.3	ns
		Titer	325.9 ± 380.0	222.8 ± 221.0	ns	245.2 ± 355.0	176.5 ± 216	ns	611.8 ± 671.0	468.1 ± 707	ns	585.3 ± 1055.8	415.2 ± 644.8	ns	214.5 ± 306.4	159.4 ± 205.0	ns
NS3	IgM	Seroc	92.3	84.6	ns	100.0	100.0	-	93.3	86.6	ns	100.0	92.8	ns	100.0	91.6	ns
		Titer	171.0 ± 428.8	33.1 ± 43.9	< 0.001	211.1 ± 300.6	62.18 ± 69.55	0.003	55.7 ± 85.7	23.6 ± 14.2	0.040	94.7 ± 146.0	132.3 ± 334.7	ns	214.3 ± 297.3	110.6 ± 192.2	ns
	IgA	Seroc	76.9	50.0	0.008	66.6	60.0	ns	66.6	26.6	0.030	57.1	42.8	ns	83.3	75.0	ns
		Titer	30.2 ± 87.6	24.1 ± 75.9	< 0.001	83.7 ± 240.4	16.3 ± 18.4	0.009	12.0 ± 7.7	8.2 ± 3.6	0.005	20.3 ± 31.8	19.8 ± 41.4	ns	47.8 ± 82.9	53.1 ± 100.2	ns
	IgG	Seroc	96.1	96.1	-	100.0	100.0	-	100.0	100.0	-	100.0	100.0	-	100.0	100.0	-
		Titer	15907.0 ± 15723.0	9684.0 ± 12663.0	0.002	11881.0 ± 7195.0	6493.0 ± 5575.0	0.001	10695.0 ± 10046.4	5166.0 ± 5304.0	0.020	8135.0 ± 8076.2	4570.0 ± 5277.1	0.030	13893.1 ± 11677.0	8806.2 ± 8510.0	ns
Neutralizing antibody response
Positive (%)			46.2	46.7	ns	66.7	58.3	ns	35.7	14.3	ns	23.1	30.8	ns	41.7	41.7	ns
Infectivity (%)			71.0 ± 77.7	58.7 ± 26.6	ns	52.6 ± 24.7	51.0 ± 20.0	ns	61.7 ± 25.1	76.1 ± 22.8	ns	73.5 ± 29.4	60.8 ± 23.8	0.040	53.2 ± 26.5	53.2 ± 26.5	ns

Seroc: Seroconversion; ns: Not significant. Seroconversion data are presented as percent of samples with detectable antibody responses. Titer and infectivity data are presented as mean ± SD, except for IgA response against the capsid in the control group, which is presented as median ± rank. Statistical values refer to time dependent (week 0 vs week 48) differences in one group (McNemar test and paired t test or Wilcoxon signed rank test). Significant differences were considered for P < 0.05. Positive (%) refers to the percent of samples with detectable neutralizing antibody response. Infectivity (%) = (foci forming units in the presence of antibodies from a patient/foci forming unit in the absence of antibodies) × 100.

Table 3 Cell-mediated immune responses

Antigen		Control group			E6 group			E9 group			L6 group			L9 group
		Week 0	Week 48	P value	Week 0	Week 48	P value	Week 0	Week 48	P value	Week 0	Week 48	P value	Week 0	Week 48	P value
Lymphoproliferative response
Capsid	Total n	25	25		14	13		15	14		13	14		12	11
	Positive (%)	4	0	ns	7.1	15.4	ns	20	14.3	ns	38.5	7.1	ns	8.3	18.2	ns
	SI1	1.2 ± 0.8	0.8 ± 0.5	ns	1.1 ± 0.6	1.2 ± 0.9	ns	1.8 ± 1.2	2.5 ± 3.4	ns	2.3 ± 2.0	1.5 ± 1.2	ns	1.5 ± 0.8	1.7 ± 1.8	ns
E2	Total n	25	23		13	9		14	10		13	10		12	9
	Positive (%)	16	8.7	ns	7.7	11.1	ns	21.4	0	ns	23.1	0	ns	13.3	0	ns
	SI	1.5 ± 1.8	1.6 ± 3.1	ns	0.9 ± 0.7	1.2 ± 1.2	ns	2.2 ± 2.2	0.6 ± 0.3	0.04	2.3 ± 2.4	1.1 ± 0.6	ns	1.4 ± 1.1	1.2 ± 0.8	ns
NS3	Total n	24	21		13	9		13	10		13	10		12	8
	Positive (%)	8.3	0	ns	7.7	11.1	ns	7.7	0	ns	30.8	0	ns	8.3	0	ns
	SI	1.2 ± 0.9	1.0 ± 0.6	ns	1.2 ± 0.8	2.0 ± 3.5	ns	1.7 ± 1.4	1.1 ± 0.4	ns	2.0 ± 1.3	0.9 ± 0.4	0.03	1.3 ± 0.8	1.1 ± 0.5	ns
IFN-γ ELISPOT response
Capsid	Total n	24	22		13	9		11	7		12	9		9	7
	Positive (%)	16.7	9.1	ns	23.1	0	ns	27.3	12.5	ns	8.3	27.3	ns	33.3	0	-
	SFC	151.4 ± 228.4	38.2 ± 67.3	0.003	232.9 ± 466.2	49.8 ± 94.1	ns	422.4 ± 731.7	50.5 ± 80.1	ns	120.9 ± 112.4	101.0 ± 89.5	ns	330.0 ± 214.7	116.0 ± 125.3	ns
E2	Total n	15	16		10	7		5	4		6	4		2	4
	Positive (%)	20	12.5	ns	10	0	-	0	0	-	0	20	ns	50	0	-
	SFC	84.6 ± 117.9	42.0 ± 82.4	0.040	96.8 ± 144.1	31.2 ± 54.8	ns	245.7 ± 409.4	3.9 ± 1.7	ns	19.9 ± 14.4	30.0 ± 33.6	ns	120.6 ± 73.9	23.3 ± 24.8	ns
NS3	Total n	17	12		9	4		1	4		7	3		4	3
	Positive (%)	15.4	8.3	ns	11.1	0	-	100	0	ns	100	0	-	25	0	-
	SFC	132.1 ± 259.7	52.0 ± 97.4	0.030	329.1 ± 452.3	2.5 ± 3.5	ns	110.8 ± 0.0	7.92 ± 10.7	-	31.3 ± 30.8	26.9 ± 23.5	ns	260.7 ± 315.7	15.2 ± 8.4	ns

Data on number of positive samples are presented as percent of total evaluated samples. Stimulation index and spot forming cells data are presented as mean ± SD. Statistical values refer to time dependent differences in one group (week 0 vs week 48; P < 0.05, Wilcoxon signed rank test).

¹A statistically significant difference regarding core-specific lymphoproliferation was observed among the groups on week 0 (P = 0.04, Kruskal-Wallis test), but the multiple comparison test was not potent enough to indicate which of the groups accounted for the difference. SI: Stimulation index; ns: Not significant; SFC: Sport forming cells/10⁶ cells.

Table 4 Leukocyte and lymphocyte counts in hepatitis C virus chronically infected patients treated with interferon-α plus ribavirin

Groups		Control group	Group E6	Group E9	Group L6	Group L9
Total number of patients		30	16	16	15	15
Baseline leukocyte counts	mean ± SD	6.7 ± 2.1	7.0 ± 1.5	7.7 ± 2.3	6.4 ± 2.0	7.0 ± 2.2
	(Min; Max)	(3.5; 12.1)	(4.2; 9.0)	(3.8; 12.2)	(4.0; 11.1)	(2.0; 10.5)
Week 12 leukocyte counts	mean ± SD	4.5 ± 1.5	4.4 ± 1.8	5.0 ± 1.5	3.7 ± 0.9	4.7 ± 0.9
	(Min; Max)	(2.3; 9.0)	(2.3; 8.4)	(3.1; 7.3)	(2.7; 5.1)	(3.4; 6.8)
Week 48 leukocyte counts	mean ± SD	4.3 ± 1.3	3.6 ± 1.2	4.4 ± 1.5	3.7 ± 1.3	4.9 ± 0.9
	(Min; Max)	(2.5; 7.6)	(1.6; 6.7)	(2.3; 7.6)	(2.5; 6.6)	(2.9; 5.8)
Statistics (P value)1		< 0.001	0.001	< 0.001	0.001	0.013
Statistics (P value)2		< 0.010	0.001	0.001	0.001	0.023
Baseline lymphocyte counts	mean ± SD	37.8 ± 8.6	35.7 ± 7.6	33.7 ± 7.5	36.7 ± 10.7	37.6 ± 8.7
	(Min; Max)	(22.8; 58.8)	(21.8; 52.5)	(19.8; 53.8)	(18.3; 51.4)	(25.4; 50.7)
Week 12 lymphocyte counts	mean ± SD	38.2 ± 9.7	34.6 ± 6.9	30.5 ± 8.0	36.1 ± 9.5	36.5 ± 7.5
	(Min; Max)	(20.3; 52.9)	(22.0; 44.1)	(15.8; 47.4)	(22.5; 55.2)	(25.3; 52.0)
Week 48 lymphocyte counts	mean ± SD	32.0 ± 8.9	37.1 ± 8.3	31.2 ± 6.9	34.9 ± 11.3	33.1 ± 6.2
	(Min; Max)	(13.3; 57.8)	(19.5; 50.9)	(21.3; 45.0)	(19.8; 54.0)	(23.5; 42.3)
Statistics (P value)1		ns	ns	ns	ns	ns
Statistics (P value)2		0.004	ns	ns	ns	ns

¹Week 12 vs baseline in one group;

²Week 48 vs baseline in one group (Differences were considered significant when P < 0.05; Wilcoxon signed ranks test for leukocyte counts, Paired t test for lymphocyte counts). Results are given as 10⁹ cells/L in the case of leukocytes and as % in the case of lymphocytes. ns: Not significant.

Table 5 CIGB-230 administration induces differential courses of cell-mediated immunity in virological responder and non-responder patients under therapy with interferon-α plus ribavirin

Immune response		HCV RNA (-) on week 48			HCV RNA (+) on week 48			HCV RNA (-) on week 72			HCV RNA (+) on week 72
		Week 0	Week 48	P value	Week 0	Week 48	P value	Week 0	Week 48	P value	Week 0	Week 48	P value
CIGB-230 immunized patients
IgG	Core	32193.3	15486.7	0.0001	22068.7	15130.5	ns	32193.3	15486.7	0.003	22920.0	15130.5	0.0041
	E2	272.3	103.5	0.0061	50	295.3	ns	138.8	103.1	ns	254.3	138.7	ns
	NS3	8277.2	2969.1	< 0.0001	7177.4	5555.5	0.006	8013.1	2900.1	0.002	7263.8	5555.5	0.0002
IFN-γ	Core	69.2	15	ns	88.8	57.5	ns	68.8	18.3	ns	76.3	43.5	0.041
	E2	17.4	5.6	ns	10.8	5.8	ns	22.5	5.2	ns	10.8	5.8	ns
	NS3	14.6	2.5	ns	38.8	16.3	ns	15	0	ns	26.3	15.1	ns
SI	Core	1.3	1	ns	1.4	1	ns	1.3	1	0.04	1.4	1	ns
	E2	1	0.9	0.0061	0.9	1.2	ns	1	0.8	ns	0.9	1.2	ns
	NS3	1.2	1	ns	0.9	0.9	ns	1.2	1.1	ns	1.1	0.9	ns
Control patients
IgG	Core	26820.1	9096.3	0.0010	14778.9	10761.0	0.008	28066.9	12800.0	0.002	14778.9	8873.2	0.0005
	E2	85.7	233.1	ns	339.3	94.5	0.040	85.7	185	ns	339.3	155	ns
	NS3	5150.3	2856.7	0.0010	15003.7	10833.1	ns	5150.3	2856.7	0.003	15003.8	8577.9	ns
IFN-γ	Core	78.7	19.1	0.0410	60.3	15.6	ns	80.4	35	ns	60.3	14	0.0200
	E2	39.5	6.6	ns	5	0	ns	36.2	7.5	ns	32.5	2.5	ns
	NS3	35.9	4.1	0.0310	11.6	ne	-	47.7	13	ns	12.9	4.2	ns
SI	Core	0.9	0.8	ns	1.2	0.8	ns	1	0.7	ns	1.1	1	ns
	E2	0.8a	0.8	ns	1.8a	1.4	ns	0.8c	0.7	ns	1.4c	1.4	ns
	NS3	0.8	0.8	ns	1.5	1.1	ns	0.8	0.7	ns	1.2	1.1	ns

Median values are shown. SI: Stimulation index; ns: Not significant; ne: Not evaluated. Differences were considered statistically significant when P < 0.05 (Wilcoxon signed rank test).

¹Not significant when virological relapsers and non-responders were separately analyzed. ^a,cIndicate differences between virological responders and non responders, on both evaluation time points (week 48 and week 72) (P < 0.05, Unpaired t test). HCV: Hepatitis C virus.