Preclinical evaluation of a Haemophilus influenzae type b conjugate vaccine process intended for technology transfer

Figures & data

Table 1. Summary of injection site reactions and body weights at day 63 (sacrifice of main group) and mean food conversion efficiencies at day 77 (total study period)

Dose	Injection site reaction						Mean body weights (d63) (g) ± sem		Mean food conversion efficiency^2) ± sem (d0-d77)
	Erythema			Haematoma
	1	2	3	1	2	3	♂	♀	♂	♀
Placebo	0	2	0	0	0	1	381.0 ± 10.3	234.0 ± 4.6	0.12 ± 0.01	0.06 ± 0.01
Test Hib Vaccine	0	4	1	0	0	1	394.5 ± 9.3	221.7 ± 3.9	0.12 ± 0.02	0.05 ± 0.01
Licensed Hib Vaccine	0	3	0	0	0	2	377.2 ± 9.1	222.9 ± 4.1	0.12 ± 0.01	0.06 ± 0.01

Abbreviations: d, day; sem, standard error of the mean. (1) Number of animals showing the observation for both male (♂) and female (♀) rats. (2) Gram weight gain/g food consumed.

Table 2. Summary of main haematology and clinical chemistry data

		Placebo					Test Hib vaccine					Licensed Hib vaccine
Time point		d1	d29	d57	d63	d77	d1	d29	d57	d63	d77	d1	d29	d57	d63	d77
Haematology	N m/f	8/8	9/9	9/9	9/9	3/3	9/9	9/9	9/9	9/9	3/3	8/9	9/9	9/9	9/9	3/3
Total white blood cells (*10^9/L ± sem)	♂	9.7 ± 0.6	11.5 ± 0.6	12.2 ± 0.4	10.2 ± 0.06	10.8 ± 1.5	9.0 ± 0.6	11.7 ± 0.5	12.5 ± 0.7	11.3 ± 0.5	10.6 ± 0.6	10.2 ± 1.3	13.4* ± 0.5	13.8 ± 0.4	12.3* ± 0.4	10.6 ± 1.1
	♀	6.8 ± 0.7	10.1 ± 0.4	9.2 ± 0.8	6.6 ± 0.5	6.9 ± 0.6	6.5 ± 0.3	9.3 ± 0.5	8.4 ± 0.5	5.6 ± 0.4	7.0 ± 0.3	7.0 ± 0.5	9.1 ± 0.4	8.4 ± 0.5	6.0 ± 0.5	6.3 ± 0.7
Eosinophils (% ± sem)	♂	0.5 ± 0.3	0.6 ± 0.2	1.6 ± 0.3	1.0 ± 0.3	1.3 ± 0.9	0.2 ± 0.1	2.2** ± 0.5	1.4 ± 0.3	1.7 ± 0.6	1.0 ± 0.6	0.3 ± 0.2	1.2 ± 0.3	0.7 ± 0.4	0.2 ± 0.1	1.0 ± 0.6
	♀	0.8 ± 0.3	1.2 ± 0.7	2.0 ± 0.3	0.9 ± 0.4	0.7 ± 0.7	0.2 ± 0.1	0.7 ± 0.4	1.6 ± 0.3	0.8 ± 0.3	1.7 ± 1.2	1.3 ± 0.5	2.0 ± 0.5	2.1 ± 0.4	0.7 ± 0.3	1.0 ± 1.0
Neutrophils (% ± sem)	♂	8.3 ± 1.7	5.7 ± 1.0	6.0 ± 0.8	7.2 ± 0.9	8.0 ± 3.0	7.4 ± 0.9	6.4 ± 0.9	7.9 ± 2.2	5.2 ± 0.6	7.0 ± 1.7	11.6 ± 1.3	8.1 ± 2.0	6.4 ± 0.6	8.7 ± 1.7	10.7 ± 1.8
	♀	6.0 ± 0.3	8.3 ± 1.1	5.9 ± 1.7	10.0 ± 1.5	9.0 ± 3.1	7.6 ± 1.0	6.8 ± 1.3	8.9* ± 1.0	8.8 ± 1.3	14.3 ± 6.4	4.9 ± 0.9	7.7 ± 1.2	5.3 ± 0.9	8.3 ± 1.9	6.7 ± 2.2
Lymphocytes (% ± sem)	♂	88.3 ± 1.3	91.6 ± 0.8	90.9 ± 0.7	89.7 ± 1.1	88.0 ± 2.6	91.0 ± 0.9	89.2 ± 1.3	88.6 ± 2.1	91.9 ± 0.9	90.0 ± 1.0	87.1 ± 1.3	88.8 ± 2.2	91.1 ± 1.0	90.1 ± 1.7	86.7 ± 1.5
	♀	92.8 ± 0.4	89.7 ± 1.5	91.2 ± 1.9	88.3 ± 1.7	89.0 ± 3.2	91.0 ± 1.1	91.0 ± 1.3	87.8** ± 0.9	89.3 ± 1.3	83.3 ± 5.8	92.0 ± 0.9	89.0 ± 1.4	90.8 ± 1.2	90.3 ± 1.8	91.0 ± 2.5
Monocytes (% ± sem)	♂	3.0 ± 0.5	2.1 ± 0.4	1.6 ± 0.4	2.1 ± 0.6	2.7 ± 0.3	1.3* ± 0.4	2.0 ± 0.5	2.1 ± 0.5	1.2 ± 0.4	2.0 ± 0.6	1.0** ± 0.4	1.9 ± 0.4	1.8 ± 0.4	1.0 ± 0.3	1.7 ± 0.9
	♀	0.5 ± 0.2	0.8 ± 0.2	0.9 ± 0.3	0.8 ± 0.4	1.3 ± 0.9	1.2 ± 0.3	1.6 ± 0.4	1.8 ± 0.4	1.1 ± 0.5	0.7 ± 0.7	1.8 ± 0.6	1.3 ± 0.3	1.8 ± 0.5	0.7 ± 0.3	1.3 ± 0.7
Clinical chemistry	n	9/9	9/9	9/9	9/9	3/3	9/9	9/9	9/9	9/9	3/3	9/9	9/9	9/9	9/9	3/3
Albumin/Globulin ratio (± sem)	♂	1.72 ± 0.03	1.68 ± 0.02	1.58 ± 0.02	1.55 ± 0.04	1.74 ± 0.06	1.64 ± 0.03	1.62 ± 0.06	1.36** ± 0.03	1.49 ± 0.02	1.59 ± 0.05	1.55** ± 0.03	1.49* ± 0.04	1.45** ± 0.03	1.53 ± 0.02	1.58 ± 0.04
	♀	1.97 ± 0.04	2.00 ± 0.06	1.99 ± 0.05	2.00 ± 0.05	2.31 ± 0.05	1.82* ± 0.04	1.91 ± 0.04	1.68** ± 0.04	1.97 ± 0.04	1.97** ± 0.04	1.87 ± 0.03	1.99 ± 0.04	1.85 ± 0.06	20.9 ± 0.04	2.27 ± 0.07

Statistics: (Two-Sided) Anova + Dunnett tests, *P ≤ 5%; **P ≤ 1% (both bold). Abbreviations: d, day; N m/f, Number of rats male/female; sem, standard error of the mean; P, probability value.

Figure 1. PRP- specific IgG antibodies in serum after vaccination. Rats were vaccinated with 3 doses of a licensed Hib vaccine (square) and the test Hib vaccine (triangle) on day 0, 28, and 56. As a negative control a group of rats was vaccinated using a placebo (circle). The level of PRP- specific IgG antibodies were measured in a rat-anti-PRP ELISA at OD450

Table 3. Study design of the repeated dose toxicity study with Haemophilus influenza b vaccine

Parameter	Signs looked for		Days
Clinical observations	- Erythema - Haematoma - Edema - Body weights - Food intake - Skin changes		0–63 At least ones daily
Haematology	- hemoglobin - packed cell volume - red blood cell count - reticulocytes - thrombocyte count - total white blood cell count - differential white blood cell count	- Prothrombin time - mean corpuscular volume - mean corpuscular hemoglobin - mean corpuscular hemoglobin concentration	1, 29, 57, 63, (77)
Clinical Chemistry	- alkaline phosphate activity - alanine aminotransferase activity - aspartate aminotransferase activity - gamma glutamyl transferase activity - total protein - albumin - ratio Albumin to Globulin - urea	- creatinine bilirubin - Cholesterol - triglycerides - phospholipids - calcium - sodium - potassium - chloride - inorganic phosphate	1, 29, 57, 63, (77)
Pathology	Isolation, macroscopy, organ weight		The day of necropsy (63 or 77)
Histopathology	- spleen - thymus - bone and bone marrow - muscle at the injection sites(hind legs) and untreated muscle (left fore leg) - draining popliteal lymph nodes - draining, inguinal lymph nodes - gross lesions		The day of necropsy (63 or 77)