Evaluation of humoral and cellular immune responses to a DNA vaccine encoding chicken type II collagen for rheumatoid arthritis in normal rats

Figures & data

Table 1. The plasma levels of anti-CII antibodies from vaccinated normal rats with pcDNA-CCOL2A1 vaccine at different time-points after a single intramuscular injection of therapeutic dosage of 300 ug/kg (Units/mL, n = 30).

Anti-CII antibodies	NC^1	3d	7d	10d	14d	21d	28d	35d
Rat-IgG Anti-Rat	<0.000	<0.000	<0.000	<0.000	<0.000	<0.000	<0.000	<0.000
Rat-IgG Anti-Chicken	<0.000	<0.000	<0.000	<0.000	<0.000	<0.000	<0.000	<0.000

1. NC: normal control. Each plasma was not diluted, and values were expressed as mean units of activity.These data are epresentative of 3 experiments. Three separate experiments yielded similar results.

Table 2. The plasma concentrations of inflammatory and anti-inflammatory cytokines from vaccinnated normal rats with pcDNA-CCOL2A1 vaccine at different time-points after a single intramuscular injection of therapeutic dosage of 300μg/kg(pg/mL, n = 30).

Cytokines concentrations	IL-1α	IL-5	IL-6	IL-12	G-CSF	MIP-1α	MCP-1
NC^1	5.02–17.88	45.79–144.42	2.22–7.35	119.58–299.94	18.22–42.63	2.66–9.72	237.80–767.31
3d	5.20–18.68	55.13–122.62	2.49–7.92	117.86–270.81	18.86–45.52	2.15–10.42	245.82–751.90
7d	5.92–19.42	45.53–135.66	2.03–7.42	111.98–283.88	18.64–48.12	2.53–11.53	254.46–791.21
10d	5.07–16.94	40.33–123.50	1.84–6.27	117.52–305.09	17.96–56.94	2.63–9.52	236.20–998.56
14d	5.53–18.17	49.95–139.12	1.83–7.16	116.33–284.26	18.62–60.27	1.89–9.59	228.13–469.81
21d	5.41–29.13	38.72–126.41	1.75–7.73	117.75–297.22	18.11–43.01	1.56–10.96	303.55–769.17
28d	5.40–32.08	43.52–145.57	1.84–7.65	121.93–379.13	18.09–44.34	2.33–10.30	343.05–619.08
35d	5.81–24.49	55.17–149.28	1.95–8.09	109.00–310.55	17.89–47.52	2.52–10.11	253.37–525.54
Cytokines concentrations	RANKL	RANTES	TNF-α	IFN-γ	IL-4	IL-10	TGF-β
NC	27.25–132.71	12.22–51.7	4.70–8.40	1.61–3.78	3.30–8.49	10.30–29.96	1874.93–5346.13
3d	34.12–135.87	13.08–58.01	5.10–7.96	1.12–3.79	3.54–8.88	11.48–30.85	1865.52–4826.58
7d	39.52–137.23	11.60–59.20	5.05–8.31	1.42–3.86	2.57–8.42	10.28–23.86	2469.81–5762.07
10d	37.94–135.12	13.26–61.18	5.05–11.65	1.22–7.21	2.09–8.69	13.55–51.09	4538.61–12779.28*
14d	24.31–91.23	10.59–46.89	5.28–13.53	0.91–2.52	3.25–8.94	10.54–30.92	4545.51–11553.75*
21d	49.69–140.69	10.90–62.30	5.04–9.16	1.10–5.22	2.07–7.76	10.49–41.77	3027.60–6879.74
28d	57.18–140.46	14.67–53.05	—	—	3.10–9.30	18.52–39.89	2778.33–6556.48
35d	44.56–133.13	10.55–35.79	—	—	2.89–9.53	17.99–55.80	1778.58–4205.79

1. NC: normal control. Values were expressed as mean units of activity. Data represent the normal range for vaccinated normal rats. *P < 0.05 vs. the extremum of NC using the t-test when the mean of determination is outside the normal range. These data are representative of 3 experiments. Three separate experiments yielded similar results.

Table 3. The plasma concentrations of inflammatory and anti-inflammatory cytokines from vaccinnated normal rats with pcDNA-CCOL2A1 vaccine on the 14th day after a single intramuscular injection of maximum dosage of 3 mg/kg (pg/mL, n = 6).

Cytokines concentrations	MIP-1	MCP-1	RANKL	IL-10	TGF-β
NC^1,2	2.66–9.72	237.80–767.31	27.25–132.71	10.30–29.96	1874.93–5346.13
3 mg/kg DNA vaccine	5.10–8.55	297.78–563.25	47.19–99.62	10.75–29.03	3817.82–5371.38

1. NC: normal control. 2. Data from table 2 (n = 30). Values were expressed as mean units of activity. Data represent the normal range for vaccinated normal rats.

The data is representative of 3 experiments. Three separate experiments yielded similar results.

Figure 1. (See previous page). The cellular immune response to the pcDNA3.1-CCOL2A1 vaccine(300 μg/kg) in normal rats. (A) The percentages of peripheral blood Tc, Ts, CD4+CD29+, CD4+CD25+Treg, Th1/Th2, and Th17 cells in vaccinated and unvaccinated normal rats. Rats were administered a single dose of 300 μg/kg pcDNA3.1-CCOL2A1 vaccine, and immune system cells were analyzed 3 to 5 d after injection. *P < 0.05 versus normal control (NC) using t-tests. (B) Representative data from flow cytometry analysis of peripheral blood CD4+CD25+Treg, CD4+CD29+, Th1/Th17,Th2, and Tc/Ts cells from vaccinated and unvaccinated normal rats 3 to 35 d after a single intramuscular injection of 300 μg/kg pcDNA3.1-CCOL2A1. The left diagram in each group shows the representative isotype-matched control and the corresponding analyzed T-lymphocyte subsets on the right diagram. These data are representative of 3 experiments. Three separate experiments yielded similar results.

Figure 1. (Continued)

Figure 2. Cellular immune response to 3 mg/kg pcDNA3.1-CCOL2A1 vaccine in normal rats. (A) The percentages of peripheral blood Tc,Ts, and CD4⁺CD29⁺T cells in vaccinated and unvaccinated rats on day 14 after a single intramuscular injection of the vaccine. (B) The percentages of peripheral blood CD4⁺CD25⁺FoxP3⁺Treg, Th1/Th2, and Th17 cells from vaccinated and unvaccinated normal rats on day 14 after a single intramuscular injection of the vaccine. These data are representative of 3 experiments. Three separate experiments yielded similar results.