Repeated high-dose (5 × 10⁸ TCID₅₀) toxicity study of a third generation smallpox vaccine (IMVAMUNE) in New Zealand white rabbits

Figures & data

Table 1. Vaccination sites examined on rabbits vaccinated with IMVAMUNE or Saline Control. No macroscopic change related to treatment was apparent at necropsy on day 35 (n = 3).

	Treatment Group and Sex
	Male		Female
Parameters	Saline Control n=5	IMVAMUNEn = 5	Saline Control n=5	IMVAMUNE n=5
Animals euthanised on Day 10
Treated site 1
Dark area(s)	3	5	1	2
Treated Site 2
Dark area(s)	2	5	2	4
Thickened / Oedematous	0	1	0	1
Animals euthanised on Day 21
Treated Site 1
Dark area(s)	0	2	1	3
Treated Site 2
Dark area(s)	1	2	1	3
Thickened / Oedematous	0	1	0	1

Table 2. Summary of treatment-related changes in the average number of Leucocytes.

		Treatment group and sex
		Male		Female
Cell Types (mean)		Saline Control	IMVAMUNE	Saline Control	IMVAMUNE
Total white	Pretreatment	8.71	7.94	7.77	8.37
blood cells (x10^9/L)	Day 1	8.95	8.49	9.05	9.03
	Day 2	8.22	8.56	8.18	8.92
	Day 3	8.56	8.60	8.08	8.58
	Day 10	7.96	8.89	7.71	9.25
	Day 21	8.69	8.14	7.11	8.02
	Day 35	6.95	6.49	6.30	8.45*
Mean Neutrophils	Pretreatment	1.62	1.81	1.72	1.62
(x10^9/L)	Day 1	1.83	2.37*	1.86	1.74
	Day 2	1.54	2.03	1.46	1.56
	Day 3	1.78	1.87	1.64	1.61
	Day 10	1.44	1.87*	1.51	1.91
	Day 21	1.62	1.74	1.32	1.33
	Day 35	0.79	0.85	1.13	1.22
Monocytes	Pretreatment	0.12	0.17*	0.10	0.08
(x10^9/L)	Day 1	0.08	0.09	0.08	0.13**
	Day 2	0.07	0.16**	0.11	0.26*
	Day 3	0.08	0.12	0.13	0.17
	Day 10	0.17	0.17	0.06	0.08*
	Day 21	0.10	0.10	0.11	0.09
	Day 35	0.10	0.17	0.04	0.09
Lymphocytes	Pretreatment	6.30	5.31**	5.42	5.93
(x10^9/L)	Day 1	6.34	5.42*	6.61	6.46
	Day 2	5.98	5.78	6.07	6.41
	Day 3	6.04	6.02	5.76	6.10
	Day 10	5.75	6.27	5.56	6.83
	Day 21	6.31	5.75	5.22	6.08
	Day 35	5.62	5.12	4.67	6.55

Statistically significant when compared with the Control (Group 1): *-p<0.05; **-p<0.01.

Table 3. Summary of treatment-related changes in adrenal and prostate weights (g). Absolute values and difference from control (xn).

		Treatment group and sex
		Male		Female
OrganDay euthanised	No. of animals	Saline Control	IMVAMUNE	Saline Control	IMVAMUNE
Adrenals
Day 10	n = 5	0.211	0.170 (x0.81)	0.249	0.256 (x1.03)
Day 21	n = 5	0.234	0.233 (x1.00)	0.237	0.282 (x1.19)
Day 35	n = 3	0.167	0.285* (x1.71)	0.287	0.370 (x1.29)
Prostate
Day 10	n = 5	0.797	0.603 (x0.76)	–	–
Day 21	n = 5	0.683	0.503 (x0.74)	–	–
Day 35	n = 3	0.597	1.115 (x1.87)	–	–

Statistically significant when compared with the Saline Control (Group 1): *-p<0.05

Figure 1. Mean vaccinia-specific IgG titer (Log₁₀ ± 1 SE) of New Zealand white rabbits vaccinated with MVA-BN (IMVAMUNE) vaccine (Group 2). Animals were bled on various days (Prebleed Day 0, Day 7 after the first immunization, 3 d (Day 10), 14 d (Day 21) and 28 d (Day 35) after the second immunisation. Mean values are from 13 (Day 0 and Day 7), 5 (Day 10 and Day 21) or 3 (Day 35) animals each. LOD = Limit of Detection (1.7 log₁₀). First immunization (▴), Second immunisation (▪). For graphical purposes values below the limit of detection (LOD) were assigned a value of 1Log_10.

Table 4. Summary of treatment-related changes in the axillary lymph nodes on days 10, 21 and 35. Number of animals with treatment related changes/Total number of animals.

Prominent germinal centers	Treatment group and sex
	Male		Female
	Saline Control	IMVAMUNE	Saline Control	IMVAMUNE
Day 10
Minimal	0/5	0/5	0/4	3/5
Slight	0/5	2/5	0/5	1/5
Day 21
Minimal	0/5	4/5	0/5	4/5
Day 35
Minimal	0/3	3/3	0/3	3/3

Table 5. Study Plan: Animals were vaccinated subcutaneously twice (7-days apart) with either MVA-BN (IMVAMUNE) (Group 2), or with saline control (Group 1). Animals were monitored for 27 d (upto day 35) after the last vaccination. Animals were euthanised at days 10, 21 and 35.

			Number of animals
			Euthanised Day 10		Euthanised Day 21		Euthanised Day 35
Group No.	Treatment	Dose volume(ml)	Male	Female	Male	Female	Male	Female
1	Saline Control	2×1ml	5	5	5	5	3	3
2	IMVAMUNE	2×1ml^a	5	5	5	5	3	3

^a One dose of IMVAMUNE consisted of 2 × 0.5ml equivalent to 4.9×10⁸ TCID₅₀.