Optimization and qualification of an assay that demonstrates that a FimH vaccine induces functional antibody responses in women with histories of urinary tract infections

Figures & data

Figure 1. A. Effect of centrifugation on fluorescence. Assays were performed as described in the Methods section, except that T24 cell numbers were varied as indicated, and appropriately diluted E. coli was added directly to the adhered T24 cells, without a pre-incubation with test or control serum or antibodies. For each T24 cell number, two plates were prepared: one was centrifuged as described in the Methods, and one was not. Plotted here is the ratio of fluorescence from the centrifuged plate to fluorescence from the non-centrifuged plate. N = 2 wells per condition, and error bars represent standard deviation. B. Fluorescence signal as a function of CFU/well. Assays were performed as described in the Methods section, except that appropriately diluted E. coli was added directly to the adhered T24 cells, without a pre-incubation with test or control serum or antibodies. N = 6 or 12 wells per condition, and error bars represent standard deviations. C. Inhibition of fluorescence by mannose with varied CFU E. coli/well. Assays were performed as described in the Methods section, with a range of mannose concentrations. N = 2 wells per condition, and error bars represent standard deviation. D. Inhibition of fluorescence by mannose or rabbit anti-FimH. Assays were performed as described in the Methods section, with varied incubation time of E. coli with inhibitor. N = 6 (30 min and 120 min incubations) or 12 wells (controls, 60 min inubations) per condition, and error bars represent standard deviation. Two replicate plates were run on two separate days; data from one representative plate are shown

Figure 2. A. Inhibition of binding by rabbit serum. Serum was collected from a single rabbit before and 4 weeks after immunization with the FimCH vaccine. The assay was performed as described in the Methods section. Low CFU wells contained 3 × 10⁶ CFU/well; all others contained 6 × 10⁶ CFU/well. Two replicate plates were run on two separate days; data from one representative plate are shown. N = 6 wells per condition, and error bars represent standard deviation. The RSDs of the average fluorescence signals on each plate were ≤12%. B. Inhibition of binding by purified human anti-FimH IgG. Low CFU wells contained 3 × 10⁶ CFU/well; all others contained 6 × 10⁶ CFU/well. Two replicate plates were run on two separate days; data from one representative plate are shown. N = 6 wells per condition, and error bars represent standard deviation. The RSDs of the average fluorescence signals were <12%. C. Effect of heat inactivation of serum on fluorescence. Assays were performed as described in the Methods section, except that heat inactivation time varied. Low CFU wells contained 3 × 10⁶ CFU/well, and mid CFU wells contained 4.5 × 10⁶ CFU/well; all others contained 6 × 10⁶ CFU/well. Serum taken from one subject before and after vaccination were assayed after 0, 1, or 2 h heat inactivation. N = 6 wells per condition, and error bars represent standard deviation

Figure 3. Inhibition of E. coli binding by anti-FimH sera from immunized humans with a history of urinary tract infections. Pre-immune sera were collected prior to the first vaccination, and post-immune sera were collected 30 days after the fourth vaccination. Serum from subjects 101–6004, 102–6005, and 103–6001 were each tested on two separate days as noted in the graph titles. N = 6 wells per condition, and error bars represent standard deviation

Figure 4. Plate Map 1 (upper) and Plate Map 2 (lower). Each plate contains Serum from a single patient plus bacterial controls and mannose controls

Table 1. Fluorescence as a function of concentration of rabbit anti-FimH IgG RS (reference standard) spiked into a pool of normal human sera. The full range of spiking concentrations were measured across two assay plates

Plate 1 Sample ID	Average Fluorescence (N = 6)	%RSD
Low CFU Control	26615	5.9
Intermediate CFU Control	36114	6.2
High CFU Control	44415	5.3
Mannose Positive Control	28467	6.9
Non-Spiked NHS	44282	5.5
300 µg/mL Anti-FimH IgG RS	22167	6.6
150 µg/mL Anti-FimH IgG RS	23070	6.9
75 µg/mL Anti-FimH IgG RS	24361	4.8
37.5 µg/mL Anti-FimH IgG RS	27599	5.9
Plate 2 Sample ID
Low CFU Control	27605	1.4
Intermediate CFU Control	37100	2.9
High CFU Control	46163	2.7
Mannose Positive Control	29433	3.4
Non-Spiked NHS	47318	3.5
18.8 µg/mL Anti-FimH IgG RS	38009	2.4
9.4 µg/mL Anti-FimH IgG RS	44644	2.5
4.7 µg/mL Anti-FimH IgG RS	47415	2.2
2.3 µg/mL Anti-FimH IgG RS	47121	2.2

Table 2. Selectivity and Specificity; rabbit anti-FimH IgG spiked at 100 µg/mL into a range of dilutions of NHS

Sample ID	Total Number of Replicates	Average Fluorescence	Standard Deviation	%RSD	Positive/Negative for Inhibition
Low CFU Control	12	26971	796.8	2.8%	Positive
Intermediate CFU Control	12	36760	1290.5	3.4%	N/A
High CFU Control	12	46021	1799.3	3.7%	Negative
Mannose Positive Control	12	33016	2317.0	6.7%	Positive
1:10 NHS; Non-Spiked	6	47893	1472.1	2.8%	Negative
1:20 NHS; Non-Spiked	6	47100	1596.3	3.1%	Negative
1:40 NHS; Non-Spiked	6	47108	2540.2	4.9%	Negative
1:10 NHS; Spiked	6	32036	1684.1	4.8%	Positive
1:20 NHS; Spiked	6	32086	2010.8	5.7%	Positive
1:40 NHS; Spiked	6	30429	1888.8	5.7%	Positive
1:80 NHS; Spiked	6	28810	1744.6	5.5%	Positive
1:160 NHS; Spiked	6	29656	1979.6	6.1%	Positive
1:320 NHS; Spiked	6	29970	1707.8	5.2%	Positive
1:640 NHS; Spiked	6	28949	1924.9	6.1%	Positive
T24 Only	12	8640	170.7	1.9%	N/A

Figure 5. Fluorescence as a function of concentration of rabbit anti-FimH IgG RS (reference standard) spiked into a pool of normal human sera. Individual values are shown in Table 1

Table 3. Intra-Assay Precision; average, SD, and RSD across replicate wells within a single plate

Run ID	Control ID	Replicates	Average Fluorescence	SD	RSD
Run 1	HPC	6	34569	1320.4	3.8%
	LPC	6	37471	2052.8	5.5%
	NC	6	49713	1403.8	2.8%
Run 2	HPC	6	30613	2738.3	8.9%
	LPC	6	34609	3360.7	9.7%
	NC	6	46939	3481.8	7.4%
Run 3	HPC	6	20765	653.6	3.1%
	LPC	6	26010	922.6	3.5%
	NC	6	46524	1272.0	2.7%

Table 4. Inter-Assay Precision; average, SD, and RSD of results from three different plates

Control ID	Run ID	Average Fluorescence	Inter-Assay Mean	SD	RSD
HPC	Run 1, Day 1	34569	28649	7108.6	24.8%
	Run 2, Day 2	30613
	Run 3, Day 3	20765
LPC	Run 1, Day 1	37471	32697	5965.4	18.2%
	Run 2, Day 2	34609
	Run 3, Day 3	26010
NC	Run 1, Day 1	49713	47725	1733.8	3.6%
	Run 2, Day 2	46939
	Run 3, Day 3	46524

Table 5. Dilutions of Sera from FimH-Immunized Humans that Inhibit the Adherence of E. coli to Human Bladder Cells

Vaccine Administered	Subject Number	Sera Dilutions that Inhibited the Adherence of ≥1.5 x 10^6 CFU/well of E. coli
		Day 1	Day 210
50 µg of FimCH with 40 µg of PHAD®	101–06-001	<10	40
	101–06-004	<20	40
	101–06-005	<10	10
	102–06-004	10	320
	103–06-001	<20	40
	105–06-002	<10	20
	106–06-001	10	160
	102–06-005	<20	320
	101–06-007	<10	20
	106–06-002	<10	80
	106–06-003	<20	20
	106–06-004	no sample	160
	101–06-008	10	640
107 µg of FimCH with 43 µg of PHAD®	106–07-001	<10	<10
	106–07-002	<10	160
	106–07-003	<10	160
	102–07-001	<10	160
	105–07-002	<10	20
	107–07-003	<10	80
	105–07-003	<10	40
	105–07-005	<10	40
	106–07-004	<10	20
	106–07-006	<10	160
	106–07-007	<10	20
	101–07-004	<10	10
	106–07-008	<10	80