The cationic liposome CCS/C adjuvant induces immunity to influenza independently of the adaptor protein MyD88

Figures & data

Table 1. Proliferative response and cytokine production by spleen cells following two doses of F-HA vs. CCS/C-HA vaccination in WT, Tlr2-/-, and Tlr4-/- mice

		Spleen
Vaccine (n = 5)	Incubation Treatment	Proliferation (SI^a)	IFN γ (pg/ml)	IL-2 (pg/ml)	IL-5 (pg/ml)
Con A	0.25 µg HA/ml	8.2	>1000	70 ± 13	152 ± 21
PBS	0.5 µg HA/ml	1.4	0	3 ± 1	0
	0.05 µg HA/ml	1.5	0	2 ± 0.4	0
F-HA WT	0.5 µg HA/ml	2	182 ± 10	75 ± 28	439 ± 79
	0.05 µg HA/ml	1.5	154 ± 18	57 ± 20	391 ± 62
CCS/C-HA WT	0.5 µg HA/ml	4	400 ± 22	96 ± 21	660 ± 59
	0.05 µg HA/ml	2.4	422 ± 65	22 ± 13	620 ± 28
F-HA Tlr2^−/-	0.5 µg HA/ml	1.8	0	63 ± 11	547 ± 28
	0.05 µg HA/ml	1.8	0	53 ± 13	368 ± 25
CCS/C-HA Tlr2^−/-	0.5 µg HA/ml	3.2	593 ± 62	590 ± 29	292 ± 45
	0.05 µg HA/ml	5	581 ± 112	342 ± 29	217 ± 25
F-HA Tlr4^−/-	0.5 µg HA/ml	0.8	29 ± 18	85 ± 17	325 ± 57
	0.05 µg HA/ml	1	0	61 ± 6	200 ± 23
CCS/C-HA Tlr4^−/-	0.5 µg HA/ml	3.3	417 ± 26	316 ± 38	306 ± 41
	0.05 µg HA/ml	2.6	463 ± 18	318 ± 42	302 ± 35

C57BL/6 (WT), Tlr2^−/- and Tlr4^−/- female mice (n = 3–5) were vaccinated on d 0 and 28 with PBS only or F-HA vs. CCS/C-HA (Aventis Pasteur Vaxigrip 2008–09, 3 μg HA). Spleen cells were harvested 38 d after the first vaccination (10 d after boost). ^aSI>2, positive.

Table 2. Proliferative response and cytokine production by spleen cells following vaccination with one or two doses of F-HA vs. CCS/C-HA vaccine in WT and Myd88-/- mice

		Spleen
		Proliferation (SI^a)		IFN γ (pg/ml)		IL-2 (pg/ml)		IL-5 (pg/ml)
Vaccine (n = 5)	Incubation Treatment	Group A	Group B	Group A	Group B	Group A	Group B	Group A	Group B
Con A	0.25 µg HA/ml	5.1	3.3	>1000	>1000	10 ± 3.5	40 ± 6	215 ± 11	95 ± 16
PBS	0.5 µg HA/ml	1.1	1.1	0	0	0	0	0	0
	0.05 µg HA/ml	1.4	1.4	0	0	0	0	0	0
F-HA WT	0.5 µg HA/ml	0.3	0.4	3313 ± 279	2068 ± 216	25 ± 3.5	33 ± 3.5	97 ± 30	494 ± 38
	0.05 µg HA/ml	0.3	0.6	1680 ± 382	825 ± 35	20 ± 1	30 ± 7	68 ± 9	403 ± 16
CCS/C-HA WT	0.5 µg HA/ml	3.8	3	1103 ± 138	9640 ± 105	130 ± 14	225 ± 7	0	39 ± 8
	0.05 µg HA/ml	2.4	1.8	325 ± 21	1585 ± 219	88 ± 18	133 ± 11	0	0
F-HA Myd88^−/-	0.5 µg HA/ml	0.5	0.6	0	0	15 ± 3.5	68 ± 11	15 ± 3.5	258 ± 30
	0.05 µg HA/ml	1.1	1.2	0	0	0	35 ± 11	0	82 ± 21
CCS/C-HA Myd88^−/-	0.5 µg HA/ml	2.1	2.7	1040 ± 113	2615 ± 290	413 ± 17	188 ± 18	299 ± 28	988 ± 58
	0.05 µg HA/ml	2.8	3	480 ± 40	1895 ± 290	253 ± 11	150 ± 14	188 ± 23	836 ± 40

C57BL/6 (WT) and Myd88^−/- female mice (n = 3), were vaccinated once (Group A) or twice (d 0 and 27, Group B) with PBS only, or F-HA vs. CCS/C-HA (Aventis Pasteur Vaxigrip 2008–09, 3 μg HA). Spleen cells were harvested 30 d (Group A) and 36 d (Group B, 9 d after boost) after vaccination. ^aSI>2, positive.

Figure 1. Effect of mouse strain and gender on immunogenicity of F-HA vs. CCS/C-HA

HI titer following F-HA vs. CCS/C-HA i.m. immunization of different strains of female mice, and Balb/c male mice (all age matched, n = 5). Mice were immunized with 0.5 µg HA/mouse in a volume of 50 µl. Serum HI titer was tested 4 weeks after immunization against A/Solomon Islands/3/2006 (H1N1) virus and is shown as mean ±SD. *P ≤ 0.05, ns, not significant. ANOVA across strains indicated a highly significant effect of treatment with CCS/C-HA vs. F-HA (P < .0001).

Figure 2. TLR2 and TLR4 do not contribute to antiviral immunity induced by F-HA and CCS/C-HA

HI titer following one immunization (A) or two immunizations (B) with F-HA vs. CCS/C-HA in WT, TLR2 KO, and TLR4 KO mice. Mice (n = 6) were immunized with 1 µg HA/mouse of each viral strain (3 µg total) in a volume of 50 µl. The CCS/C formulation contained 0.3 mg lipid/mouse. Serum HI titers against A/Brisbane (H1N1) virus were tested individually 26 d after the first vaccination and 10 d after the second vaccination. For each vaccine, there were no significant differences between the responses of WT, TLR2 KO, and TLR4 KO mice. The effect of the F-HA vs. CCS/C-HA vaccine within each strain of mice in response to one or two immunizations, ***P ≤ 0.001, **P ≤ 0.01, **P ≤ 0.05, ns, not significant.

Figure 3. Adjuvant effect of CCS/C-HA does not require MyD88

HI titer following one immunization (A) or two immunizations (B) with F-HA vs. CCS/C-HA in WT, and Myd88^−/- mice. Mice were immunized with 1 µg HA/mouse of each viral strain (3 µg total) in a volume of 50 µl. The CCS/C formulation contained 0.3 mg lipid/mouse. Serum HI titers against A/Brisbane virus were tested individually 26 d after the first vaccination and 9 d after the second vaccination. Percentages shown represent seroconversion rates defined by HI titer ≥40.