Intranasal vaccination with an inactivated whole influenza virus vaccine induces strong antibody responses in serum and nasal mucus of healthy adults

Figures & data

Figure 1. HI antibody responses in serum. (A) HI antibody responses before and after primary and secondary vaccination are shown for serum. A paired t test was performed to compare data from week 0 (pre) and 6 (post). Correlation coefficient (r) and p value were calculated. *; p < 0.05. (B) The relationship of serum HI antibody responses before and after secondary vaccination. The abscissa and ordinate show the pre- and post-vaccination HI titers, respectively. Further, it is shown how these relate to conversion rate and protection rate, which are on the border or within the area marked by the bold line and by the light gray background, respectively. Each circle represents an individual and shows the relation between the pre- and post-vaccination titers. Gray circles indicate subjects between 60 and 69 y-of-age.

Table 1. Tools currently in use to evaluate vaccine-induced changes in serum HI titer

Name	Description	EMA criteria ^a	FDA criteria ^b
Mean geometric increase	Ratio of the geometric mean titer (GMT) post vaccination to that pre-vaccination	> 2.5	No standard
Conversion rate or significant increase in titer [n/N (%, 95% CI)] ^c	Conversion rate: Proportion of subjects showing an increase from a pre-vaccination titer of < 1:10 (non-immune) to a post-vaccination titer of ≥ 1:40 (designated immune state). Significant increase in titer: Proportion of subjects showing a 4-fold or greater increase from a pre-vaccination titer of ≧ 1:10 (designated as a significant increase in titer).	> 40%	LL of 95% CI > 40% ^d
Protection rate [n/N (%, 95% CI)] ^c	Proportion of subjects showing a post-vaccination titer of ≧ 1:40	> 70%	LL of 95% CI > 70% ^e

^a European Medicines Agency (EMA) criteria for serum HI antibody responses in people aged 18–60 y. ^b US Food and Drug Administration (FDA) criteria for serum HI antibody responses in adults < 65 y. ^c n = the number of subjests who meet a requirement; n = total number of subjects; 95% CI = the 95% Confidence Interval. ^d Lower limit (LL) of the 95% CI should exceed 40%. ^e Lower limit (LL) of the 95% CI should exceed 70%.

Table 2. Serum HI antibody responses after two doses of the nasal A/Victoria vaccine

	EMA criteria	FDA criteria	Week	Serum ^a
GMT (Mean geometric increase)	>2.5	No standard	0	16.2 (1.00)
			6	68.8 (4.25 ^b)
Conversion rate or Significant increase in titer [n/N (%, 95% CI)]	>40%	LL of 95%CI > 40%	6	20/46 (43.5, 28.5–58.4)
Protection rate [n/N (%, 95% CI)]	>70%	LL of 95%CI > 70%	0	13/46 (28.3, 14.7–41.8)
			6	35/46 (76.1, 63.2–88.9)

^a Serum HI titers of <1:10 were considered negative and were arbitrarily assigned a titer of 1:5. ^b In bold values that exceed EMA criteria as defined for HI titers.

Figure 2. Neutralizing antibody responses and correlation between HI and NT titers in serum. (A) Neutralizing antibody responses before and after primary and secondary vaccination are shown. A paired t test was performed to compare data from week 0 (pre) and 6 (post). The correlation coefficient (r) and p value were calculated. **; p < 0.01. (B) Correlation between HI and NT titers in serum 3 weeks after the secondary nasal vaccination. The abscissa and ordinate show HI and NT titers, respectively. Pearson r value and p value were calculated. Each individual is represented by a circle showing corresponding HI and NT titers. Gray circles indicate subjects between 60 and 69 y-of-age.

Table 3. Serum neutralizing antibody responses after two doses of the nasal vaccine

	Time after first vaccination (week)	Serum ^a
GMT (Mean geometric increase)	0	28.7 (1.00)
	6	229.7 (8.00)
Conversion rate or significant increase in titer [n/N (%, 95% CI)] ^b	6	29/46 (63.0, 48.5–77.6)
Protection rate [n/N (%, 95% CI)] ^b	0	14/46 (30.4, 16.6–44.3)
	6	40/46 (87.0, 76.8–97.1)

^a Serum NT titers of <1:10 were considered negative and were arbitrarily assigned a titer of 1:5. ^b Conversion rate and protection rate were tentatively estimated by correcting for the relative ratio between GMT of NT titers and that of HI titers; a serum NT titer of 1:80 was considered to be a 1:40 of the lowest protection level in HI titers.

Figure 3. HI and neutralizing antibody responses in nasal mucus. HI (A) and NT (B) titers before and after primary and secondary vaccination. A paired t test was performed to compare data from week 0 (pre) and 6 (post). The correlation coefficient (r) and p value were calculated. *; p < 0.05, ***; p < 0.001. (C) Correlation between HI and NT titers in nasal wash 3 weeks after the secondary nasal vaccination. The abscissa and ordinate show HI and NT titers, respectively. Pearson r value and p value were calculated. Each circle represents an individual showing corresponding HI and NT titers. Gray circles indicate subjects between 60 and 69 y-of-age.

Table 4. Nasal HI and neutralizing antibody responses after two doses of the nasal vaccine

	Week	HI titers ^a	NT titers ^a
GMT (Mean geometric increase)	0	12.4 (1.00)	15.4 (1.00)
	6	38.8 (3.13)	90.5 (5.88)

^a Nasal HI and neutralization titers were determined using concentrated nasal wash samples containing 1 mg/ml total protein. Nasal HI and neutralization titers of <1:20 were considered negative and were arbitrarily assigned a titer of 1:10.

Table 5. Serum and nasal HA-specific antibody responses after two doses of the nasal vaccine

	Isotype	Week	Serum	Nasal wash
GMT (Mean geometric increase)	IgG	0	83.7 (1.00)	80.0 (1.00)
		6	247.7 (2.96)	109.8 (1.37)
	IgA	0	14.6 (1.00)	122.0 (1.00)
		6	36.0 (2.47)	473.5 (3.88)

^a Serum HA-specific antibody titers of < 1:10 were considered negative and arbitrarily assigned a titer of 1:5. ^b Nasal HA-specific antibody responses were determined using concentrated nasal wash samples containing 1 mg/ml total protein. Nasal HA-specific IgG and IgA titers of < 1:160 were considered negative and arbitrarily assigned a titer of 1:80.

Table 6. Cross-reactive HI and neutralizing antibody responses to A/Sydney virus after two doses of the A/Victoria vaccine

Item	Antibody responses	Week	Serum^a	Nasal wash^b
GMT (Mean geometric increase)	HI antibody	0	21.2 (1.00)	13.6 (1.00)
		6	30.5 (1.44)	22.2 (1.63)
	Neutralizing antibody	0	160.0 (1.00)	23.0 (1.00)
		6	233.2 (1.46)	48.7 (2.12)

^a Serum HI and neutralization titers of < 1:10 were considered negative and arbitrarily assigned a titer of 1:5. ^b Nasal wash HI and neutralization titers were determined using concentrated nasal wash samples containing 1 mg/ml total protein. Nasal HI and neutralization titers of < 1:20 were considered negative and arbitrarily assigned a titer of 1:10.