Universal H1N1 influenza vaccine development

Figures & data

Figure 1. Informatic-driven identification of influenza H1N1 hemagglutinin and neuraminidase immunogenic consensus sequences. Results of the step-wise computational process of screening H1-HA and N1-NA sequences for conserved and potentially immunogenic epitopes and constructing immunogenic consensus sequences are shown.

Figure 2. Conservation distribution of influenza H1N1 hemagglutinin and neuraminidase 9-mers. Pre-pandemic and pandemic H1-HA (A) and N1-NA (B) sequences were parsed into overlapping nine amino acid frames with a one amino acid frameshift, and unique 9-mers were evaluated for percent coverage of source antigens at 100% sequence identity using the Conservatrix algorithm. Inset: Close-up of the 75–100% coverage range.

Figure 3. Immunogenic consensus sequence influenza H1N1 strain coverage and HLA binding potential. ICS construction yielded 13 H1-HA and four N1-NA sequences with > 85% coverage of input proteins and EpiMatrix cluster scores > 10, representing significant HLA binding potential. Bars represent EpiMatrix cluster score and open circles percent strain coverage.

Table 1. H1N1 immunogenic consensus sequences

Hemagglutinin Immunogenic Consensus Sequences								IEDB		% Coverage
#	HA POS	NTERM	CORE PEPTIDE	CTERM	# Hits	CLUSTER SCORE	# EpiBars	HLA Binding	T Cell Assay	All H1N1	Pandemic H1N1	Pre-Pandemic H1N1
										2273 Unique Sequences	1410 Unique Sequences	870 Unique Sequences
1	112	FIQ	YEELREQLSSVS	AFE	9	14.78	1		Y	98.7%	98.65%	97.01%
2	440	AEM	LVLLENERTLDY	YDS	10	16.02	1	Y	Y	99.2%	98.94%	99.20%
3	523	ESA	RIYQILAIYSTVAS	TLV	15	25.59	3		Y	99.6%	99.01%	99.20%
4	396	VNS	IIEKMNTQFTAVG	KEF	8	11.07	1	Y	Y	98.9%	98.44%	99.20%
5	527	IYQ	ILVIYSTVASSLVL	SVS	20	35.15	4		Y	98.6%	98.09%	99.20%
6	463	YEK	VRSQLKNNAKEIG	NGC	12	22.54	2		Y	99.0%	96.31%	98.51%
7	548	GAV	SFWMCSNGSLQ	FRI	7	11.78	1		Y	97.4%	95.82%	99.66%
8	432	FMD	IWTYNAELLVLL	DNE	7	10.15	1	Y	Y	99.7%	99.29%	100.00%
9	38	ERN	VTVTHSVNLLE	EKH	8	13.13	1		Y	95.1%	92.41%	98.97%
10	19	TLS	IGYHANNSTDT	EDT	6	10.35	1		Y	99.7%	99.57%	99.54%
11	326	STR	LRMVTGLRNVPSIQS	KGL	17	28.36	3	Y	Y	93.3%	91.99%	93.33%
13	386	QNA	INRITNKVNSVI	KKM	8	11.54	2		Y	96.8%	98.23%	94.48%
19	337	NVP	FIQSRGLFGAIAGFI	EGG	12	19.73	2	Y	Y	99.5%	99.22%	99.08%
Neuraminidase Immunogenic Consensus Sequences								IEDB		% Coverage
#	NA POS	NTERM	CORE PEPTIDE	CTERM	# Hits	CLUSTER SCORE	# EpiBars	HLA Binding	T Cell Assay	All H1N1	Pandemic H1N1	Pre-Pandemic H1N1
										2273 Unique Sequences	1410 Unique Sequences	870 Unique Sequences
1	129	CRN	FFLTQGALLN	DRH	12	23.64	2		Y	98.4%	99.11%	97.83%
2	79	SVL	SVKLAGNSSLCS	TSG	12	22.78	2	Y	Y	89.2%	92.14%	82.79%
3	17	IIG	LILQIGNIISI	WPV	14	27.31	2		Y	93.2%	96.59%	89.70%
4	439	SGS	IISFCGVNSDTV	DWS	7	11.19	1		Y	80.6%	95.40%	65.45%

Key features of HA and NA ICS produced by the EpiAssembler algorithm are presented, including position in the native antigen sequence relative to Influenza A/California/04/2009 (HA/NA POS), N- and C-terminal flanking residues (NTERM/CTERM), core immunogenic peptide sequence (CORE PEPTIDE), EpiMatrix 9-mer-to-HLA-allele hits (#HITS), overall peptide promiscuous binding potential where ≥ 10 is significant (CLUSTER SCORE), number of 9-mers with promiscuous binding potential where ≥ 1 is significant (#EpiBars). Peptides previously shown to bind HLA-DR (IEDB: HLA Binding) or to stimulate HLA-DR-restricted T cell responses (IEDB: T Cell Assays) as reported in the Immune Epitope Database are noted. Conservation is summarized by classification (Pandemic vs. Pre-Pandemic) and overall. % Coverage describes how many of the individual protein isolates are represented with at least one of the HLA-binding 9-mers in an ICS, as opposed to the average conservation of each of the constituent 9-mers in an ICS core peptide across the total set of protein isolates (Avg 9-mer % Cons).

Figure 4. ICS peptide – HLA DR binding affinities. Peptide identifiers and sequences are noted in the first and second columns, respectively. IC₅₀ values in μM units were calculated from curves fitted to dose-dependence competition binding data for each peptide-HLA DR allele pair. Peptide binding affinity is shown according to the following classification: IC₅₀ < 0.1 µM (black), 0.1 µM < IC₅₀ < 1 µM (dark gray), 1 µM < IC₅₀ < 10 µM (gray), 10 µM < IC₅₀ < 100 µM (light gray), IC₅₀ > 100 µM (lightest gray). IC₅₀ values too high to accurately measure under binding conditions tested are considered non-binders (NB; shown in white cells).

Figure 5. Antigen-specific human IFNγ ELISpot responses to computationally identified influenza HA and NA immunogenic consensus sequences. ICS were assayed for T cell reactivity by IFNγ ELISpot assay using PBMCs isolated from normal human donors (n = 5). An ELISpot response was considered positive if three criteria were met: (1) spot-forming cells (SFC) per million PBMC were at least 50 over background; (2) SFC per million PBMC were at least 2-fold over background; and (3) antigen-stimulated SFC numbers were statistically different (p < 0.05) from non-stimulated counts. (A) The numbers of SFC over background per million PBMCs that secrete IFNγ in response to individual and pooled influenza HA and NA ICS are presented. Individual subject responses are represented by dots and the average response across subjects by horizontal lines. The 50 SFC over background per million PBMCs cutoff is denoted by the dotted line. (B) The ELISpot response stimulation index, representing the ratio of antigen-stimulated SFC counts to non-stimulated counts, is presented. Stimulation index values per individual subject are represented by dots and the average values across subjects by horizontal lines.

Table 2. Cultured human IFNγ ELISpot responses to influenza H1N1 ICS peptides

	844 (DR1, DR4)			1489 (DR1, DR4)			1003 (DR4, DR4)			720 (DR1, DR7)			1442 (DR4, DR13)			% Subjects Positive
	SFC	SI	p-value	SFC	SI	p-value	SFC	SI	p-value	SFC	SI	p-value	SFC	SI	p-value
HA-1	143	1.30	0.346	267	1.23	0.129	397	5.17	< 0.001	293	11.00	0.001	507	0.85	0.217	40%
HA-2	137	1.24	0.506	207	0.95	0.741	177	2.30	0.002	310	11.63	< 0.001	540	0.91	0.327	40%
HA-3	117	1.06	0.863	603	2.78	< 0.001	1180	15.39	< 0.001	280	10.50	< 0.001	2297	3.87	< 0.001	80%
HA-4	137	1.24	0.529	610	2.82	0.005	1000	13.04	< 0.001	587	22.00	< 0.001	1023	1.72	< 0.001	60%
HA-5	97	0.88	0.711	170	0.78	0.171	70	0.91	0.733	103	3.88	< 0.001	480	0.81	0.042	20%
HA-6	120	1.09	0.797	120	0.55	0.035	30	0.39	0.036	13	0.50	0.121	687	1.16	0.116	0%
HA-7	150	1.36	0.301	163	0.75	0.143	107	1.39	0.142	57	2.13	0.184	760	1.28	0.029	0%
HA-8	153	1.39	0.231	810	3.74	< 0.001	1217	15.87	< 0.001	163	6.13	0.034	1277	2.15	< 0.001	80%
HA-9	133	1.21	0.521	210	0.97	0.856	160	2.09	0.003	43	1.63	0.063	530	0.89	0.365	20%
HA-10	190	1.73	0.081	263	1.22	0.301	147	1.91	0.008	53	2.00	0.032	513	0.87	0.185	20%
HA-11	203	1.85	0.080	820	3.78	< 0.001	837	10.91	< 0.001	177	6.63	0.001	777	1.31	0.034	60%
HA-13	110	1.00	1.000	190	0.88	0.529	97	1.26	0.316	50	1.88	0.036	497	0.84	0.071	0%
HA-19	83	0.76	0.477	170	0.78	0.154	3763	49.09	< 0.001	73	2.75	0.028	3643	6.14	< 0.001	60%
NA-1	140	1.27	0.390	200	0.92	0.580	127	1.65	0.147	143	5.38	0.009	630	1.06	0.470	20%
NA-2	143	1.30	0.346	230	1.06	0.740	270	3.52	< 0.001	117	4.38	< 0.001	517	0.87	0.226	40%
NA-3	147	1.33	0.318	260	1.20	0.279	200	2.61	0.006	33	1.25	0.553	537	0.90	0.494	20%
NA-4	150	1.36	0.270	210	0.97	0.873	103	1.35	0.385	27	1.00	1.000	580	0.98	0.795	0%
ICS Pool	223	2.03	0.026	2457	11.34	< 0.001	4813	62.78	< 0.001	1153	43.25	< 0.001	3971	6.69	< 0.001	100%

Immunoreactivity data for influenza H1N1 ICS peptides are presented from cultured IFNγ ELISpot assays performed for five human subjects. An ELISpot response was considered positive if three criteria were met: (1) spot-forming cells (SFC) per million PBMC were at least 50, (2) SFC per million PBMC were at least 2-fold over background, and (3) SFC per million PBMC were statistically different from “no stimulus” measurements by the Student’s t-test (p < 0.05). Results that meet individual criteria are highlighted in gray. Results that meet all three criteria are denoted in bold. Column headers: human subject ID code and HLA class II type in parentheses; numbers of spot forming cells per million PBMCs that secrete IFNγ in response to individual and pooled ICS peptides (SFC); stimulation indices (SI); and Student’s t-test results (p-value); and percent of subjects responding. Row labels: peptide ID. Epitopes are grouped according to their source antigen, either HA or NA. It is possible Subject 844 did not respond strongly to the ICS peptides because of no prior history of H1N1 exposure or vaccination.