The compatibility of inactivated-Enterovirus 71 vaccination with Coxsackievirus A16 and Poliovirus immunizations in humans and animals

Figures & data

Table 1. Clinical trials for 3 human Enterovirus 71 (EV71) vaccines

Clinical trials	Institute	Time	Place	Age	Group	ClinicalTrials.gov Identifier
1	Beijing Vigoo Biological Co., LTD	January 2011	Jiangsu, China	13-60 m, 6-12 m	160U, 320U, 640U vaccine and Placebo	NCT01313715
2	Sinovac Biotech Co., Ltd	December 2010	Guangxi, China	3–11 y, 6–35 m	100U, 200U, 400U vaccine and Placebo	NCT01273246
3	Institute of Medical Biology, CAMS	February 2011	Guangxi, China	18–49 y, 3–11 y, and 6–35 m	160Eu, 320Eu and Placebo	NCT01391494
4	Beijing Vigoo Biological Co., LTD	August 2011	Jiangsu, China	6–12 m, 13–36 m	160U, 320U, 640U vaccine and Placebo	NCT01399853

Table 2. The change of EV71 and CA16 NTAbs in infants and children in clinical trial 1, 2 and 3

			EV71 NTAb				CA16 NTAb
			Seropositive ratio		GMTs (95%CI)				Seropositive ratio		GMTs (95%CI)
Clinicaltrial	Group	Total number	Pre	Post	Pre	Post	P	Seroconversionratio	GMFI	Pre	Post	Pre	Post	P	Seroconversion ratio	GMFI
Clinical trial 1	640 U/Dose	9	5/9	9/9	26.9 (6.5～111.8)	4019.4 (2548.5～6339.2)	<0.001	9/9	149.3 *	4/9	4/9	11.4 (3.3～39.6)	22.1 (4.5～109.0)	0.289	2/9	1.9
	320 U/Dose	8	5/8	8/8	57.5 (8.9～371.0)	1109.4 (295.0～4172.9)	0.002	8/8	19.3	5/8	5/8	27.7 (7.2～106.4)	64.9 (8.4～502.1)	0.056	2/8	2.3
	160 U/Dose	10	8/10	10/10	79.7 (23.7～268.6)	1525.9 (559.2～4163.8)	<0.001	10/10	19.1	9/10	9/10	53.9 (18.1～160.1)	65.5 (19.6～219.2)	0.5	1/10	1.2
	Placebo	11	7/11	8/11	76.1 (9.6～605.2)	63.7 (10.2～396.6)	0.652	1/11	0.8 *	8/11	9/11	39.1 (9.7～157.7)	58.3 (15.1～225.3)	0.411	2/11	1.5
	P		>0.05	<0.05	<0.01	<0.0001		<0.01	<0.0001	>0.05	>0.05	>0.05	>0.05		>0.05	>0.05
Clinical trial 2	400 U/Dose	6	4/6	6/6	29.7 (4.2～207.7)	6762.4 (985.3～46413.2)	<0.001	6/6	228.1*	0/6	0/6	4 (4～4)	4 (4～4)	/	0/6	1.0
	200 U/Dose	11	1/11	11/11	5.3 (2.8～10.2)	222.2 (61.8～799.5)	<0.001	11/11	41.6	3/11	3/11	9.5 (3.4～26.3)	15.2 (3.1～73.8)	0.181	2/11	1.6
	100 U/Dose	9	1/9	9/9	4.7 (3.3～6.7)	208.6 (56.4～771.6)	<0.001	9/9	44.7	2/9	2/9	8.1 (2.8～23.8)	7.7 (2.8～21.3)	0.347	0/9	1.0
	Placebo	8	0/8	0/8	4 (4～4)	4 (4～4)	/	0/8	1.0 *	0/8	0/8	4 (4～4)	4 (4～4)	/	0/8	1.0
	P		<0.05	<0.01	>0.05	<0.0001		<0.01	<0.0001	>0.05	>0.05	>0.05	>0.05		>0.05	>0.05
Clinical trial 3	320 Eu/Dose	10	7/10	10/10	35.1 (7.1～174.2)	886.8 (194.5～4044.0)	<0.001	9/10	25.3	3/10	5/10	9.3 (3.1～28.3)	26.3 (5.2～133.8)	0.061	4/10	2.8
	160 Eu/Dose	9	2/9	9/9	10.5 (2.4～46.8)	93.5 (15.5～563.1)	<0.001	7/9	8.9	2/9	3/9	6 (3.1～11.6)	11 (2.9～41.4)	0.198	2/9	1.9
	Placebo	10	6/10	5/10	27.4 (6.1～122.2)	46.3 (7.1～302.2)	0.266	1/10	1.7 *	1/10	4/10	4.5 (3.5～5.7)	15.5 (4.2～57.1)	0.051	4/10	3.5
	P		>0.05	<0.01	>0.05	<0.05		<0.01	<0.001	>0.05	>0.05	>0.05	>0.05		>0.05	>0.05

P value is the comparison result for each category in each clinical trial. Seropositivity is defined as NTAb titers equal to or greater than 1:8. Seroconversion is defined with at least 4-fold increase on the post-vaccination titer compared to the pre-vaccination titer. GMT = geometric mean titer. GMFI = geometric mean fold increase.

*:There were significant differences with other groups in that clinical trial.

Table 3. The change of EV71 and Poliovirus NTAbs in infants and children from clinical trial 4

			Seropositive ratio		GMTs (95%CI)
NTAb	Group	Number	Pre	Post	Pre	Post	P	Seroconversion ratio	GMFI
EV71	640 U/Dose	20	4/20	20/20	8.6 (4.1～18.1)	691.7 (403.4～1185.9)	<0.001	20/20	80.2
	320 U/Dose	20	4/20	20/20	8.1 (3.8～17.0)	714.2 (354.8～1437.7)	<0.001	20/20	88.2
	160 U/Dose	20	2/20	20/20	6.1 (3.3～11.0)	689.9 (351.5～1354.0)	<0.001	19/20	113.8
	Placebo	20	6/20	7/20	11.9 (4.8～29.0)	18.2 (5.9～56.2)	0.285	1/20	1.5*
	P		>0.05	<0.01	>0.05	<0.0001		<0.01	<0.0001
Poliovirus I	640 U/Dose	20	20/20	20/20	2037.4 (1476.2～2811.8)	1748.3 (1080.3～2829.2)	0.281	1/20	0.9
	320 U/Dose	20	20/20	20/20	1917.1 (1082.2～3396.3)	2766.8 (1564.9～4891.7)	0.016	2/20	1.4*
	160 U/Dose	20	20/20	20/20	2020.1 (1215.5～3357.3)	1287.4 (797.4～2078.3)	0.004	0/20	0.6
	Placebo	20	19/20	20/20	1229 (520.9～2899.4)	1069.9 (440.8～2597.0)	0.462	0/20	0.9
	P		>0.05	/	>0.05	>0.05		>0.05	<0.01
Poliovirus II	640 U/Dose	20	20/20	20/20	689.9 (441.7～1077.5)	492 (275.3～879.2)	0.043	0/20	0.7
	320 U/Dose	20	20/20	20/20	689.9 (421.3～1129.6)	761 (424.7～1363.4)	0.531	2/20	1.1
	160 U/Dose	20	20/20	20/20	494.8 (306.3～799.1)	380.5 (243.1～595.5)	0.094	0/20	0.8
	Placebo	20	20/20	20/20	545.9 (312.4～953.9)	519.8 (282.6～956.1)	0.802	0/20	1.0
	P		/	/	>0.05	>0.05		>0.05	>0.05
Poliovirus III	640 U/Dose	20	20/20	20/20	205.1 (131.6～319.6)	150.5 (96.4～235.0)	0.071	1/20	0.7
	320 U/Dose	20	20/20	19/20	298.5 (181.1～492.0)	282.6 (145.7～548.0)	0.830	4/20	0.9
	160 U/Dose	20	20/20	19/20	216.7 (132.6～354.2)	128.4 (72.0～229.2)	0.005	0/20	0.6
	Placebo	20	20/20	19/20	205.6 (112.5～375.9)	149.7 (74.5～300.4)	0.062	0/20	0.7
	P		/	>0.05	>0.05	>0.05		>0.05	>0.05

P value is the comparison result for each category in each clinical trial. Seropositivity is defined as NTAb titers equal to or greater than 1:8. Seroconversion is defined with at least 4-fold increase in post-vaccination titer compared to the pre-vaccination titer. GMT = geometric mean titer. GMFI = geometric mean fold increase.

*:There are significant differences with other groups in that clinical trial.

Table 4. The seropositive ratios of NTAb for inactivated-EV71 vaccination after CA16 or Poliovirus vaccination

		Seropositive ratio
Group	Time for NTAb test	EV71 NTAb*	CA16 NTAb*	Poliovirus I NTAb*	Poliovirus II NTAb*	Poliovirus III NTAb*
CA16	Pre-EV71	0/10	10/10	0/10	0/10	0/10
	Post-EV71	10/10	9/10	0/10	0/10	0/10
Poliovirus	Pre-EV71	0/10	0/10	10/10	10/10	10/10
	Post-EV71	10/10	0/10	9/10	10/10	10/10
Adjuvant	Pre-EV71	0/10	0/10	0/10	0/10	0/10
	Post-EV71	10/10	0/10	0/10	0/10	0/10
Negative control	/	0/10	0/10	0/10	0/10	0/10

*: There are significant differences of seropositive ratios among these groups（P < 0.05）.

“Pre” and “Post” are equal to before (28 day) and after (56 day) EV71 vaccination.

Figure 1. The NTAb GMTs for inactivated-EV71 vaccination after CA16 or Poliovirus vaccination. BALB/c mice (n = 10 per group) were subcutaneously injected with CA16 virus (VR18, genebank no：JX481738, 4.8 × 10⁵ PFU/mouse), inactivated-Poliovirus vaccine (Sanofi Pasteur, lot：G0510-1, Type I 20 DU/mouse, Type II 4 DU/mouse, Type III 16 DU/mouse), or aluminum adjuvant (Adjuvant). All animals were boosted in week 3 after priming. One week after the boost, all mice took the first inactivated-EV71 vaccination (SINOVAC BIOTECH CO.,LTD., 200 U/mouse), followed by the second inactivated-EV71 vaccination 3 weeks later. Negative control group was just inoculated with saline. Sera were collected on day 28 (7 days after the 2nd boost) and day 56. All the sera were stored at −20°C. Neutralization titers (NTs) of the sera for EV71 NTAb, CA16 NTAb and Polio I, II, III NTAb were determined. Data were expressed as means ± SEM. “Pre” and “Post” are equal to before (on 28 day) and after (on 56 day) EV71 vaccination. For analysis of GMTs, the data were transformed using the log 2 of the original values. Panels a-e separately show CA16, Polio I-III and EV71 neutralization titers for each group before and after EV71 vaccination and panel f shows immunization design for this experiment. Note: * means the GMTs were significantly different after vaccination (P < 0.05).*** means the GMTs were very significantly different after vaccination (P < 0.0001).

Table 5. The Seropositive ratio of NTAb for inactivated-EV71 vaccine co-immunized with CA16 or Poliovirus

	Seropositive ratio
Group	EV71 NTAb*	CA16 NTAb*	Poliovirus I NTAb*	Poliovirus II NTAb*	Poliovirus III NTAb*
EV71	10/10	0/10	0/10	0/10	0/10
CA16	0/10	10/10	0/10	0/10	0/10
PV	0/10	0/10	10/10	10/10	10/10
EV71&CA16	10/10	10/10	0/10	0/10	0/10
EV71&PV	10/10	0/10	10/10	10/10	10/10
Adjuvant	0/10	0/10	0/10	0/10	0/10

*: There are significant differences of seropositive ratios among these groups（P < 0.05).

Figure 2. The NTAb GMTs for inactivated-EV71 vaccine co-immunized with CA16 or Poliovirus vaccine. 60 pathogen-free BALB/c mice (6–8 weeks，female，purchased from Vital River Lab Animal Technology Co., Ltd, Beijing, China) were used. BALB/c mice (n = 10 per group) were subcutaneously injected with inactivated-EV71 vaccine (SINOVAC BIOTECH CO.,LTD., 200 U/mouse), CA16 virus (VR18, genebank accession no：JX481738, 4.8 × 10⁵ PFU/mouse), inactivated-Poliovirus vaccine (Sanofi Pasteur, lot：G0510-1, Type I 20 DU/mouse, Type II 4 DU/mouse, Type III 16 DU/mouse), or co-immunized with inactivated-EV71 vaccine (EV71 & CA16 group and EV71 and polio group). The control group was just inoculated with aluminum adjuvant (Adjuvant). The animals were boosted in week 3 after priming. All the sera were collected one week after the boost and stored at −20°C. Neutralization titers (NTs) of the sera were determined for EV71 NTAb, CA16 NTAb and Polio I, II, III NTAb. Data were expressed as means ± SEM. For analysis of GMTs, the data were transformed using the log 2 of the original values. Panels a-e separately show EV71, CA16 and Polio I-III neutralization titers for each group, and panel f shows immunization design for this experiment. Note: *** means this group were significantly different when compared with other groups without *** label (P < 0.0001).

Table 6. Homology of 2 EV71 neutralizing epitopes with CA16 and poliovirus

	VP2-28(VP2:136–150)	SP2(VP1:145 -159)
EV71	AGGTGTEDSHPPYKQ	EVVPQLLQYMFVPPG
CA16	AGGTGNENSHPPY	ELVPQLLQYMYVPPG
Polio I	SHHLYK	QIMYVPPG
Polio II	AGQASTEGDS	QIMYIPPG
Polio III	SHHLYK	QIMYIPPG