. 2022 Apr 6;8(14):eabl5209. doi: 10.1126/sciadv.abl5209

Table 2. Comparison of mutations arising within epitope regions in NGS datasets and NCBI consensus sequences.

Mutations within HA, NA, and NP BALB/c epitopes are compared across datasets from PBS-vaccinated [d3 (n = 5), d5 (n = 5), and d7 (n = 9)], SQIIV-vaccinated [d7 (n = 4)], and Wyeth/IL-15/5Flu–vaccinated [d7 (n = 6)] viral lung isolates after H1N1 challenge. These sequences are compared to mutations within the same epitope regions from an NCBI dataset of randomly selected sequences (n = 1000). Mutable positions are highlighted in red, and overall dominant mutation within dataset is noted as WT amino acid–epitope position-mutated amino acid. * in NA_79–93 indicates an “amber” stop mutation. N/A, not applicable.

Protein/epitope/ T cell restriction and dominance			Positions mutable				Overall mutational frequency in dataset				Dominant mutant
	Source	Epitope	NCBI	PBS	S-QIIV	Wyeth/ IL-15/5Flu	NCBI	PBS	S-QIIV	Wyeth/ IL-15/5Flu	NCBI	PBS	S-QIIV	Wyeth/ IL-15/5Flu
HA
HA_126–137	d3 p.i. H1Ca04	`SFERFEIFPKT`		2	1	1		1	0.2	0.1		F8V	F8V	F8V
CD4⁺ BALB/c	d5 p.i. H1Ca04	`SFERFEIFPKT`		2	1	1		0.8	0.5	0.3		F8V	S1P	F8V
Subdominant	d7 p.i. H1Ca04	`SFERFEIFPKT`		1	1	3		0.2	0.3	4.6		F8V	F8V	E6G

	NCBI database	`SFERFEIFPKT`	7				2.9				T11E
HA_140–154	d3 p.i. H1Ca04	`AKSFYKNLIWLVKKG`		0	0	0		0	0	0		N/A	N/A	N/A
CD4⁺ BALB/c	d5 p.i. H1Ca04	`AKSFYKNLIWLVKKG`		2	2	0		1	1.3	0		I9M	S3G	N/A
Immunodominant	d7 p.i. H1Ca04	`AKSFYKNLIWLVKKG`		0	1	4		0	0.1	5.5		N/A	L11R	N7D

	NCBI database	`AKSFYKNLIWLVKKG`	11				29.6				S3G
HA_177–199	d3 p.i. H1Ca04	`KLSKSYINDKGK`		1	0	0		0.1	0	0		D9E	N/A	N/A
CD4⁺ BALB/c	d5 p.i. H1Ca04	`KLSKSYINDKGK`		4	1	3		1.1	0.1	0.6		S5F	K4T	D9G
	d7 p.i. H1Ca04	`KLSKSYINDKGK`		0	3	1		0	0.7	2.2		N/A	L2P	L2P
Subdominant	NCBI database	`KLSKSYINDKGK`	11				42.7				K1N/I7A/D9N/G11E
HA_518–526	d3 p.i. H1Ca04	`IYSTVASSL`		0	0	0		0	0	0		N/A	N/A	N/A
CD8⁺ BALB/c	d5 p.i. H1Ca04	`IYSTVASSL`		0	0	0		0	0	0		N/A	N/A	N/A
Immunodominant	d7 p.i. H1Ca04	`IYSTVASSL`		0	0	2		0	0	1.6		N/A	N/A	T4A

	NCBI database	`IYSTVASSL`	4				4.1				V5A
NA
NA_79–93	d3 p.i. H1Ca04	`VSGWAIYSKDNSVRI`		1	0	1		1.9	0	0.1		G3R	N/A	I15L
CD4⁺ BALB/c	d5 p.i. H1Ca04	`VSGWAIYSKDNSVRI`		2	0	1		1.3	0	6.4		G3R	N/A	W4*
Subdominant	d7 p.i. H1Ca04	`VSGWAIYSKDNSVRI`		2	2	3		4.1	0.6	4.9		S8N	W4R	I15L

	NCBI database	`VSGWAIYSKDNSVRI`	11				51.9				I13V
NA_191–201	d3 p.i. H1Ca04	`LKYNGIITDTI`		0	0	0		0	0	0		N/A	N/A	N/A
CD4⁺ BALB/c	d5 p.i. H1Ca04	`LKYNGIITDTI`		0	1	2		0	0.5	1.6		N/A	D9G	N4D
Subdominant	d7 p.i. H1Ca04	`LKYNGIITDTI`		1	1	1		4.2	0.8	0.7		I7V	I11T	I11T

	NCBI database	`LKYNGIITDTI`	9					15.6				D9E
NP
NP_39–47	d3 p.i. H1Ca04	`FYIQMCTEL`		0	0	0		0	0	0		N/A	N/A	N/A
CD8⁺ BALB/c	d5 p.i. H1Ca04	`FYIQMCTEL`		0	0	0		0	0	0		N/A	N/A	N/A
Subdominant	d7 p.i. H1Ca04	`FYIQMCTEL`		0	0	0		0	0	0		N/A	N/A	N/A

	NCBI database	`FYIQMCTEL`	6				2.1				I3V
NP_55–69	d3 p.i. H1Ca04	`RLIQNSITIERMVLS`		0	0	0		0	0	0		N/A	N/A	N/A
CD4⁺ BALB/c	d5 p.i. H1Ca04	`RLIQNSITIERMVLS`		1	0	1		0.2	0	0.5		I9L	N/A	V13A
Immunodominant	d7 p.i. H1Ca04	`RLIQNSITIERMVLS`		1	2	2		2.6	1	3.2		R1L	V13A	E10G

	NCBI database	`RLIQNSITIERMVLS`	8				15				I7L
NP_147–155	d3 p.i. H1Ca04	`TYQRTRALV`		0	0	0		0	0	0		N/A	N/A	N/A
CD8⁺ BALB/c	d5 p.i. H1Ca04	`TYQRTRALV`		0	0	1		0	0	0.1		N/A	N/A	V9G
Immunodominant	d7 p.i. H1Ca04	`TYQRTRALV`		0	0	2		0	0	2.3		N/A	N/A	T1I

	NCBI database	`TYQRTRALV`	4				0.7				Q3H
NP_218–226	d3 p.i. H1Ca04	`AYERMCNIL`		0	0	0		0	0	0		N/A	N/A	N/A
CD8⁺ BALB/c	d5 p.i. H1Ca04	`AYERMCNIL`		0	0	0		0	0	0		N/A	N/A	N/A
Subdominant	d7 p.i. H1Ca04	`AYERMCNIL`		0	2	2		0	1.1	2.9		N/A	Y2C	R4G

	NCBI database	`AYERMCNIL`	7				1.7				M5I