Preclinical evaluation of Vaxfectin^®-adjuvanted Vero cell-derived seasonal split and pandemic whole virus influenza vaccines

Figures & data

Figure 1. HI titers to TIV strains in mice and guinea pigs. CD-1 mice (A–C) or Dunkan-Hartley guinea pigs (D–F) received two injections at a 3 week interval with the indicated TIV doses with (solid bars) or without (open bars) 900 μg Vaxfectin^® (“Vax”) prepared fresh at Time 0 (open and solid black bars) or after 6 mo of storage at 2–8°C (open and solid gray bars, also denoted as “-6” on x-axis labels). The y-axis depicts the GMT (+/− 95% confidence intervals for A–C) of HI antibodies measured from serum collected at week 6 (3 weeks after the second dose). Each of the three rows represents one of the three vaccine strains used to measure HI titers, indicated above each graph.

Table 1. Geometric mean titer (GMT) ratios of hemagglutination inhibition antibodies

Strain	TIV dose (μg)	GMT ratio*- mice		GMT ratio*- guinea pigs
		Time 0	6 Months	Time 0	6 Months
A/Solomon Islands/3/06 (H1N1)	15	3.8 (1040/274)	4 (735/184)	3.2 (2032/640)	3.2 (806/254)
	7.5	3.7 (1194/320)	21 (1470/70)	6.4 (2032/320)	12.7 (2560/202)
A/Wisconsin/67/05 (H3N2)	15	2.9 (1372/470)	6.1 (3378/557)	2.2 (4064/1810)	2.0 (3225/1613)
	7.5	5.3 (2389/453)	3.5 (1689/485)	2.5 (5120/2032)	3.2 (5120/1613)
B/Malaysia/2506/04 (B)	15	2.6 (970/373)	2.6 (970/368)	5.0 (1613/320)	5.0 (806/160)
	7.5	3.0 (1470/485)	5.3 (640/121)	6.4 (2560/403)	12.7 (1613/127)

Note: *GMT Ratio, GMT of Vaxfectin^®-adjuvanted TIV/GMT of TIV alone calculated from HI titers measured from serum collected at week 6 in groups of mice or guinea pigs injected at 0 and 3 weeks with freshly-prepared formulations (Time 0) or formulations stored refrigerated for 6 mo and then injected according to a 0 and 3 week schedule. The trivalent inactivated influenza vaccine (TIV) dose corresponds to the μg of HA for each of the three strains in the vaccine.

Table 2. Time course of single radial immunodiffusion recoveries of HA in TIV

	A/Solomon Islands/3/06 (H1N1)				A/Wisconsin/67/05 (H3N2)				B/Malaysia/2506/04 (B)
Sample	0 m	1 m	3 m	6 m	0 m	1 m	3 m	6 m	0 m	1 m	3 m	6 m
30 μg/mL + Vaxfectin®	27.3	31.5	27.9	16.4	24.2	27.3	15.9	16.0	27.4	21.4	24.0	20.0
30 μg/mL	29.4	37.4	30.7	30.4	24.3	31.5	21.0	30.8	28.7	24.8	28.1	26.2
% Recovery	93%	84%	91%	54%	100%	87%	76%	52%	95%	86%	85%	76%
15 μg/mL + Vaxfectin®	15.3	17.7	16.7	10.4	12	13.8	8.6	10.6	9.8	9.5	11.3	10.6
15 μg/mL	14	19.4	17.9	14.6	7.8	16.0	9.8	18.9	10.2	11.0	14.1	10.2
% Recovery	109%	92%	93%	71%	154%	86%	88%	56%	96%	86%	80%	104%

The % recovery of each HA at the indicated doses corresponding to 15 μg and 7.5 μg in 0.5 mL injection volumes (30 μg/mL and 15 μg/mL, respectively) was calculated by dividing the single radial immunodiffusion (SRD) value (measured in millimeters) of the Vaxfectin^®-containing dose by the nonadjuvanted dose as measured at 0, 1, 3, and 6 mo (m) after formulation.

Figure 2. HI titers to different doses of TIV in mice. CD-1 mice were injected as described in Figure 1 legend with the indicated doses of TIV (0.0048–3 μg of each HA) adjuvanted with (black lines) or without (gray lines) 900 μg Vaxfectin^®. Significantly higher responses (p < 0.05) were observed for the three highest dose groups.

Table 3. Concentrations of serum IgG isotypes elicited by TIV +/− Vaxfectin®

	A/Solomon Islands/3/06 (H1N1)				A/Wisconsin/67/05 (H3N2)				B/Malaysia/2506/04
	μg/mL			HI titer	μg/mL			HI titer	μg/mL			HI titer
	lgG1	lgG2a	lgG2b		lgG1	lgG2a	lgG2b		lgG1	lgG2a	lgG2b
0.015 μg HA	0.57	0.13	0.08	5	27.46	0.13	0.08	80	0.15	0.13	0.08	5
0.015 μg HA + Vax	24.31	1.00	0.67	40	14.47	0.53	0.08	160	56.65	3.18	1.16	5
Fold increase	43	8	8	8	1	4	1	2	378	24	15	1
0.15 μg HA	3.26	0.13	0.08	20	37.54	0.13	0.08	320	1.34	0.13	0.08	5
0.15 μg HA + Vax	137.30	9.77	1.98	160	274.60	3.99	5.10	1280	137.30	9.77	2.67	320
Fold increase	42	75	25	8	7	31	64	4	102	75	33	64
1.5 μg HA	100	40	6	320	190	57	3	640	114	6	3	5
1.5 μg HA + Vax	1075	238	91	2560	1006	193	67	≥ 5120	273	142	33	≥ 5120
Fold increase	11	6	15	8	5	3	22	≥ 8	2	24	11	≥ 1024

The concentrations (in μg/mL) of HA-specific IgG1, IgG2a, and IgG2b isotypes were measured by ELISA from serum collected at week 6 following injections at 0 and 3 weeks with the indicated doses of each HA in TIV alone or with 900 μg of Vaxfectin^® (“Vax”). The fold increases were calculated by dividing the concentration obtained with Vaxfectin^®-formulated TIV by the concentration of the same dose of TIV alone for each IgG isotype. The hemagglutination inhibition (HI) antibody titers from week 6 serum pooled within each group are shown for comparison for each of the influenza vaccine strains.

Figure 3. HI titers to monovalent H5N1 vaccine in mice and guinea pigs. CD-1 mice (A) or Dunkan-Hartley guinea pigs (B) received two injections at a 3 week interval with the indicated doses of a whole virus A/Vietnam/1203/04 (H5N1) vaccine with (solid bars) or without (open bars) 900 μg Vaxfectin^® freshly prepared (Time 0, open and filled black bars) or after 6 mo of storage (open and filled gray bars).

Figure 4. Frequency of vaccine-specific IFN-γ-producing T cells. BALB/c mice received two injections at a 3 week interval of the indicated TIV doses with (solid black bars) or without (open bars) Vaxfectin^® and an ex vivo IFN-γ ELISPOT assay was used to measure the number of spot forming units (SFU) of IFN-γ T cells per 10⁶ splenocytes harvested at 8, 28, 98, and 196 d after first injection. The TIV doses included 0.015 μg (A), 0.15 μg (B), and 1.5 μg (C) per each HA or the former two for the H5N1 vaccine (D). The rare instances of open bars only (e.g., upper left panel, Day 8 TIV and Day 196 TIV) indicate the adjuvanted response did not exceed the nonadjuvanted response. The sources of antigen for recalling H5-specific responses included H5N1 and recombinant H5 HA (“rH5”). Due to the large number of treatment groups tested, each bar represents the pooled splenocytes of each group; therefore no statistical analyses were possible. The TIV data were derived from two separate studies (0.015 μg and 0.15 μg doses in one study and 1.5 μg dose in a second study).