The sequence data for the SARS-CoV-2 NSP6 protein (290 amino acids long) was retrieved from The US National Center for Biotechnology Information (NCBI) virus database. These sequences were filtered for those originating from India from the onset of this disease to August 15, 2022. The Wuhan SARS-CoV-2 virus sequence was used as the reference (accession number: YP_009724389) in the mutational analysis17.

NSP6 protein sequences were aligned using CLUSTAL Omega, an online server performing alignment using hidden Markov model profiling18, with the Wuhan-type virus sequence as the reference. Jalview, an alignment viewing software, was used to detect the differences in the NSP6 region with the accession number of the Indian SARS-CoV-2 isolates. The nonsynonymous amino acid substitutions were analyzed using the Protein Variation Effect Analyzer (PROVEAN; v1.1.3) software with a cutoff score of −2.5019. The effect of NSP6 protein mutation on its stability could be inferred from the generated score.

The model of NSP6 protein was predicted using Swiss-Model online web servers. The I-Tasser software was also used to predict the NSP6 protein model. The secondary structure of the NSP6 protein was predicted using the PSIPRED online web tool. The NSP6 protein sequences were examined using the Chou and Fasman secondary structure prediction (CFSSP) online software to detect differences in the secondary structure due to a mutation20. Secondary structure prediction tools were used to analyze alterations in the formation or loss of secondary structures.

The NSP6 protein’s physicochemical parameters were determined using ExPASy’s online ProtParam tool. The NSP6 protein’s hydrophobicity was calculated using ExPASy’s Protscale tool21.

The changes in protein stability after mutation were predicted using the I-Mutant 2.0 online program, which predicts stability in terms of the Gibbs free energy change (ΔG) values, where the negative ΔG values suggest a decrease in flexibility and positive values indicate a gain in flexibility after mutation of the wild-type protein.

The B-cell epitopes of the NSP6 protein were estimated using the Immune Epitope Database (IEDB)22 and Bepipred 2.0 server23. The IFN-inducing epitopes were predicted by IFN epitope24.

The IEDB17 Tepitool server was used to predict the T-cell binding epitope and the MHC allele. The binding of processed antigen to an MHC molecule is essential for T-cell recognition and eliciting an immune response.

The NSP6 protein’s antigenicity was predicted using the Vaxijen v2.0 server25, and its allergenic properties were determined using the AllerTOP server26.

The NSP6 protein sequences of Indian-type SARS-CoV-2 isolates from the onset of this disease to August 15, 2022, were retrieved from the NCBI database along with the first SARS-CoV-2 virus sequence. In the above period, 548 NSP6 protein sequences were submitted of Indian-type isolates. These sequences were aligned to detect variations in the NSP6 protein region of Indian-type isolates relative to a Wuhan-type isolate. Those mutations in the NSP6 region were recorded with the accession number of the isolate and the mutated base in the wild-type and mutated sequences. The NSP6 region contained 654 point mutations in Indian-type SARS-CoV-2 (Supplementary Table 1). Nine mutations were frequently observed in the NSP6 protein: T77A, V149A, L37F, T181I, V190F, L125F, M183I, and V153C (Supplementary Figure S1).

Of these 654 mutations, five (I49T, M86I, L142F, I162T, and V190I) were selected for further characterization. The five nonsynonymous amino acid substitutions showed similar effects on the structure of the NSP6 protein. The five NSP6 protein mutants showed a neutral impact at a cutoff PROVEAN score of −2.5 (Table 1).

Table 1

Amino acid substitution in the helicase region	PROVEAN score	Variation effect on protein
I49T	-1.698	Neutral
M86I	-0.427	Neutral
L142F	-1.244	Neutral
I162T	-1.474	Neutral
V190I	-0.486	Neutral

Table 2

S.No.	Accession No.	Position of mutation	Wild sequence	Mutated sequence	ΔG	Protein Stability
1.	QKY59987	49	A	T	-2.80	Decreases
2.	QKV26075	86	M	I	-1.02	Decreases
3.	QKV26087	142	L	F	-0.66	Decreases
4.	QKJ68675	162	I	T	-4.61	Decreases
5.	QKJ68699	162	I	T	-4.61	Decreases
6.	QKV25895	190	V	I	-0.94	Decreases

Table 3

Physicochemical properties	NSP6	Amino acid composition	No.	Percent composition (%)
Molecular weight	33033.69	Ala (A)	20	6.9
No. of amino acids	290	Arg (R)	9	3.1
Theoretical pI	9.11	Asn (N)	10	3.4
Instability index	22.94	Asp (D)	8	2.8
No. of negatively charged (Asp+ Glu)	11	Cys (C)	10	3.4
No. of positively charged (Arg+ Lys)	21	Gln (Q)	7	2.4
aliphatic index	111.55	Glu (E)	3	1
Grand average of hydropathicity	0.790	Gly (G)	15	5.2
Estimated half-life (mammalian reticulocytes, in vitro)	1.9 hours	His (H)	4	1.4
Atomic composition		Ile (I)	16	5.5
C	1546	Leu (L)	41	14.1
H	2378	Lys (K)	12	4.1
N	360	Met (M)	17	5.9
O	385	Phe (F)	22	7.6
S	27	Pro (P)	7	2.4
Formula	C1546H2378N360O385S27	Ser (S)	17	5.9
Total number of atoms	4696	Thr (T)	21	7.2
		Trp (Y)	17	5.9
		Val (V)	28	9.7
		Phy (O)	0	0.0
		Sec (U)	0	0.0

Table 4

S.No.	Start	End	Peptide	Length	Allergenicity	Allergenicity
1	5	9	RTIKG	5	(Probable non- antigen)	Non-allergen
2	29	34	TQWSLF	6	0.8722 (Probable antigen)	Non-allergen
3	38	41	YENA	4	(Probable non- antigen)	Allergen
4	83	89	MVYMPAS	7	-0.0550 (Probable non- antigen)	Allergen
5	95	112	MTWLDMVDTSLSGFKLKD	18	1.3163 (Probable antigen)	Non-allergen
6	131	138	VYDDGARR	8	-1.4642 (Probable non- antigen)	Non-allergen
7	152	161	VYYGNALDQA	10	0.5160 (Probable antigen)	Non-allergen
8	172	178	TSNYSGV	7	0.3020(Probable non- antigen)	Non-allergen
9	202	204	ITG	3	(Probable non- antigen)	Allergen
10	236	267	RLTLGVYDYLVSTQEFRYMNSQGLLPPKNSID	32	0.6940 (Probable antigen)	Non-allergen
11	275	286	LLGVGGKPCIKV	12	1.2988 (Probable antigen)	Non-allergen

Table 5

MHC Restriction of CTL Epitope
T-cell epitope	MHC allele	Antigenicity	Allergenicity
FAMGIIAMS	HLA-Cw*0401	1.1569 (Probable antigen)	Probable nonallergen
FAMGIIAMS	H2-Db
FAMGIIAMS	H2-Dd
FAMGIIAMS	H2-Kb
FAMGIIAMS	H2-Kd
FAMGIIAMS	H2-Ld
FAMGIIAMS	HLA-G
FAMGIIAMS	H-2Qa
FAMGIIAMS	Mamu-A*01
CVMYASAVV	HLA-A*2402	0.5448 (Probable antigen)	Probable nonallergen
CVMYASAVV	HLA-B*51
CVMYASAVV	HLA-Cw*0401
CVMYASAVV	H2-Db
CVMYASAVV	H2-Dd
CVMYASAVV	H2-Kb
CVMYASAVV	H2-Kd
CVMYASAVV	H2-Ld
CVMYASAVV	HLA-G
CVMYASAVV	H-2Qa
CVMYASAVV	Mamu-A*0
ALIISVTSN	HLA-A*0203	0.8021(Probable antigen)	Probable nonallergen
ALIISVTSN	HLA-A3
ALIISVTSN	HLA-A*0301
ALIISVTSN	HLA-B8
ALIISVTSN	HLA-Cw*0401
ALIISVTSN	H2-Db
ALIISVTSN	H2-Dd
ALIISVTSN	H2-Kb
ALIISVTSN	H2-Kd
ALIISVTSN	H2-Ld
ALIISVTSN	HLA-G
ALIISVTSN	H-2Qa
ALIISVTSN	Mamu-A*01

Table 6

Serial No.	Sequence	Method	Result	Score
1	RLTLGVYDYLVSTQEFRYMN SQGLLPPKNS	SVM based	POSITIVE	5.3556924
2	LLGVGGKPCIKV	SVM based	NEGATIVE	-0.034460869
3	ITG	SVM based	POSITIVE	1.084497
4	TSNYSGV	SVM based	NEGATIVE	-0.20288055
5	VYYGNALDQA	SVM based	NEGATIVE	-0.42078445
6	VYDDGARR	SVM based	NEGATIVE	-0.19121745
7	MTWLDMVDTSLSGFKLKD	SVM based	NEGATIVE	-0.12253481
8	MVYMPAS	SVM based	POSITIVE	0.33703218
9	YENA	SVM based	POSITIVE	0.87711292
10	TQWSLF	SVM based	POSITIVE	0.20954355
11	FAMGIIAMS	SVM based	NEGATIVE	-0.53361947
12	CVMYASAVV	SVM based	POSITIVE	0.25965934
13	ALIISVTSN	SVM based	NEGATIVE	-0.23932506

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

The ΔG values for the wild-type and mutated NSP6 proteins were calculated using the I-Mutant 2.0 online server. All mutants showed generally negative and decreasing ΔG values, suggesting a decrease in flexibility in the mutated compared to the wild-type NSP6 protein (Table 2).

The prediction of the SARS-CoV-2 NSP6 protein’s physicochemical properties showed that it comprises 290 amino acids with a molecular weight of 33033.69 Da, atom number of 4696, aliphatic index of 111.55, instability index of 22.94, and grand average of hydropathy (GRAVY) score of 0.790 (Table 3). The predicted hydropathy plot showed that its N-terminal end contains more hydrophobic residues than its C-terminal end (Figure 1).

The structure of the NSP6 protein was modeled using the I-Tasser tool since no significant model could be built with the Swiss-Model method. Therefore, the three-dimensional structures were downloaded from the I-Tasser portal (Figure 2 A). The protein model also showed the molecules in close proximity to the NSP6 protein (Figure 2 B). The Prosol server predicted the solubility of the NSP6 protein sequence as 0.4, indicating good solubility (Figure 3). Therefore, the NSP6 protein can be used for vaccine preparations.

The secondary structure of the NSP6 protein was predicted using the PsiPred server, which inferred the number and position of helix, strand, and coil structures (Figure 4 A). Secondary structures were also predicted using the CFSSP tool. The M86I point mutation showed significant secondary structure changes compared to the wild-type, whereas the other mutants did not impact the secondary structure of the NSP6 protein (Figure 4 B). Replacing methionine with isoleucine leads to the loss of a sheet structure at positions 86, 87, 88, and 89. As a sulfur-containing amino acid, methionine prefers sheet structure and is present in the hydrophobic core of a protein. Therefore, its substitution results in the loss of sheet structure. This variation in the secondary structure of the NSP6 protein can greatly impact viral replication and multiplication.

Eleven linear B-cell epitopes were identified in the NSP6 proteins. Their amino acid composition, region of occurrence, antigenicity, and allergenicity are shown in Table 4. In Figure 5, the epitopic regions are shown in yellow, and the non-epitopic regions are shown in green.

We identified three T-cell epitopes that have binding ability for different alleles. The sequences of three epitopes were FAMGIIAMS, CVMYASAVV, and ALIISVTSN (Table 5). Moreover, the IFN-inducing epitope server predicted six epitopes positive for IFN-inducing capability, including both B-cell and T-cell epitopes (Table 5). Therefore, these predicted epitopes can induce IFNs for cytokine production.

In order to become a vaccine candidate, a protein must be immunogenic and non-allergenic. The antigenicity property depends on the vaccine candidate’s ability to bind to both B-cell and T-cell receptors and induce an immune response. Tables 5 and 6 show the immunogenicity of the NSP6 protein. Allergenicity predictions for all epitopes found that only three B-cell epitopes were allergenic; the others were non-allergenic. Therefore, only the non-allergenic epitopes could be used as vaccine candidates (Table 5, Table 6).