. 2021 Jun 27;8(16):2100985. doi: 10.1002/advs.202100985

Table 2.

Peptide‐based epitope mapping and peptide vaccine development against SARS‐1 and SARS‐2

Antigen–adjuvant	Animal modelDelivery route(D, day)	Efficacy and safety	Ref.
SARS‐1 B‐ & T‐cell epitopes
HLA‐A2.1 restricted CD8⁺ T‐cell epitopes S_T1 ¹¹⁷⁴NLNESLIDL¹¹⁸² ^a) S_T2 ⁹⁴⁰ALNTLVKQL⁹⁴⁸ S_T3 ³⁴⁹VADYSVLYNS³⁵⁸ S_T4 ⁶¹³DQLTPAWRI⁶²¹ N_T1 ²⁴⁶TVTKKSAAEA²⁵⁵ M_T1 ¹⁴³IIRGHLRMAG¹⁵²	HLA‐A2.1/H2‐D^b) hMHC‐I transfected C57BL/6 mice (n = 5) HLA‐A2 is highly expressed in Asians, Africans and Caucasians	CD4⁺ T‐cell epitopes were screened by measuring peptides’ (9‐ to 10‐mer) ability to stimulate IFN‐γ production and bind to HLA2.1‐MHC‐I of DCs and protect mice against lethal infection challenge. CD4⁺ T‐cell epitopes: S_T1 and S_T2 were potent inducers of IFN‐ γ and strong binders to MHC‐1, while S_T3 & S_T4 (located in the RBD), as well as M_T1 and N_T1 epitopes stimulated IFN‐γ production from convalescent patient T‐cells and mouse CD4⁺ T‐cells, but they were mild binders to HLA2.1 MHC‐I. S_T1 was the strongest binder and inducer of IFN‐γ production. S_T1, S_T2, S_T3, and N_T1 epitopes are identical in SARS‐2, while S_T4 and M_T1 each differ by a single residue (I621V) and (M150I) from their SARS‐2 sequence.	^[ ²¹¹ ^]
HLA‐restricted memory T‐cell epitopes M_T(hC)2 ¹⁴⁷HLRMAGHSL¹⁵⁵ N_T2 ²⁶⁶TKQYNVTQAF²⁷⁵ N_T(h)3 ¹⁰¹MKDLSPRWYFYYLGT¹¹⁵ S_T(h)5 ⁵¹⁶STDLIKNQCVNFNFN⁵³⁰ S_T(h)6 ⁵⁴¹SSKRFQPFQQFGRDV⁵⁵⁵ S_T(h)7 ¹⁰⁸¹GTSWFITQRNFFSPQ¹⁰⁹⁵	N‐ and M‐protein T‐cell epitopes were identified in three convalescent patients (HLA‐I A2402, A0206, A1101, A3303, A0201, B1502, B1525, B5502, B5801, B4001, C0801, C0403, C0302, C303 & C1501), 9–11 years after SARS‐1 infection. The M_T(h)2* epitope was recognized as CD8⁺ T‐cell epitope in two patients and N_T(h)3 was recognized as CD4⁺ T‐cell epitope by two patients. N_T2 and M_T(h)2 epitopes resulted in a proliferation of IFN‐γ in vitro in 13% and 33% of CD8⁺ T‐cells, respectively. S_T(h)6, S_T(h)5, and S_T(h)6 epitopes induced CD4⁺ T‐cell IFN‐γ production from a single patient only. Each SARS‐1‐S epitope had three different residues from the SARS‐2 sequence, while M_T(h)2, N_T(h)3, and N_T2 epitopes had a single different residue (M150I), (D103E) and (Q268A), respectively. Two epitopes (S_T(h)5 and S_T(h)6) were located in the RBD.		^[ ¹²⁰ ^]
IV‐injected DCs pulsed with S_T8, S_T9, or both Recombinant Vaccinia virus (rVV)‐encoding S_T8, S_T9, or both S_T8 ⁴³⁶NYNYKYRY⁴⁴³ (K440L) S_T9 ⁵²⁵VNFNFNGL⁵³² (100%)	C57BL/6J (n = 8–16) DCs: 10⁶ cells IV (D 0) rVVs 2 × 10⁶ PFU IN (D 6) SARS‐1: (MA15) 5 × 10⁴ PFU, (IN), C (42)^b)	Long‐lived memory CD8⁺ T‐cells outlast B‐cell humoral responses by years. The IV administration of peptide pulsed DCs and IN administration of cytotoxic T‐cell epitope coated‐rVVs resulted in accumulation of antigen‐specific CD8⁺ T‐cells in lung fluid, in absence of SARS‐1‐specific CD4⁺ T‐cells or B‐cells. S_T ₉ stimulated CD8⁺ T‐cell responses higher than those of S_T ₈, but both significantly stimulated CD8⁺ T‐cells and helped in their maturation to memory and poly‐functional phenotypes in vivo in mice. After lethal challenge, T‐cells produced IFN‐γ, IL‐2, and TNF‐a, and viral titers were reduced to 10^4.5 and 10² for S_T ₈ and S_T ₉ groups compared to the control (PBS) group (10⁶ PFU/g). Immunization with S_T ₈, S_T ₉, or both resulted in 60%, 80% and 90% survival rates in mice, respectively, compared to no survivors from the control group.	^[ ¹²⁹ ^]
Early clodronate or Poly I:C [5 µg] Post‐challenge T‐cell‐specific responses S_T10 ³⁶⁶CYGVSATKL³⁷⁴ (A370P) S_T11 ⁵²¹KNQCVNFNF⁵²⁹ (LS) S_T12 ¹⁰⁶¹PAICHEGKAY¹⁰⁷¹ (E1067D, Y1071H) N_T(h)4 ³⁵³ LNKHIDAYKTFPPTEPKK³⁷⁰ (100%)	BALB/c mice (n = 3–4) H2^d HLA SARS‐1 (MA15) IN 3 × 10⁴ PFU	The early macrophage depletion group showed no histological evidence of immunopathology in mouse lungs, compared to progressive interstitial pneumonia and diffuse alveolar damage in the control (PBS) group. Early depletion of alveolar macrophages using clodronate or maturation of DCs using TLR3 ligand poly I:C resulted in 100% survival of mice after lethal challenge. In contrast, stimulation with lipopolysaccharide (LPS) or late clodronate‐induced macrophage depletion resulted in lower survival (0% and 25%, respectively); no control mice survived. Antigen‐specific CD4⁺ and CD8⁺ T‐cell stimulation showed expanded populations of IFN‐γ‐producing T‐cells and antigen‐specific populations. Activated CD8⁺ T‐cells increased 4‐12‐fold with S_T ₁₀, CD4⁺ T‐cells increased 10‐fold with N _T _(h)4 epitope stimulation and 20‐fold by poly I:C. Passive transfer of mature activated DCs to naïve mice also resulted in 100% survival (similar to poly I:C and macrophage early depletion groups), and reduction in viral titers from 10^5–8 to 10² in the lungs, compared to the control group (no survivors). However, nAb titers and subclass were not evaluated.	^[ ⁶⁴ ^]
S_B13 ⁴⁷¹ALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFEL⁵⁰³ S_B14 ⁶⁰⁴ TDVSTAIHADQLTPAWRIYSTGC ⁶²⁵ S_B15 ¹¹⁴⁶ EIDRLNEVAKNLNESLIDLQELGKYEQYC¹¹⁹¹ S_B16 ⁵⁹⁷ LYQDVNCTDVSTAIHADQLTPAWRIYSTGC ⁶²⁵ [0.5 mg/kg]	Rhesus macaque monkeys (n = 6) IM (D 0, 14, 28, 42) SARS‐1 challenge 10⁶ TCID₅₀	S_B13‐15 produced nAbs with high binding affinity (2–5 nm), while S_B16 produced Abs that enhanced viral entry into VERO E6 cells, even though the S_B16 sequence is mostly shared with S_B15 . The underlined sequences were recognized by IgG1 or IgG2a‐b mAbs from convalescent patient sera. S_B14 had the highest serological reactivity (33%) among neutralizing epitopes. A construct was prepared with four epitope copies in a dendrimer design via cysteine thiol‐acetamido linker. The vaccine combining S_B13‐15 induced IgG titers and reduced viral burden from 1.4 × 10⁵ to 7 × 10³ and the number of infected lung epithelia and macrophages were reduced by half, compared to the control group. S_B13‐16 mixture performed worse than S_B13‐15 due to viral activity enhancement of S_B16 ; S_B16 alone performed worse than the negative control group. Lung pathology was significantly better with S_B13‐15 upon challenge, indicating infection control; alveolar damage with other vaccine groups was severe.	^[ ¹⁶³ ^]
Orf_1B1 ¹⁰⁶⁵SDDYIKLNGPLTVG¹⁰⁷⁸ (K1071A & T1076K) S_B17 ³²³CPFGEVFNATKF³³⁴ (K333R) S_B18 ⁴⁶⁷CTPPALNCYWPLND ⁴⁸⁰ (LS) S_B19 ⁵⁴⁵FQPFQQFGRDVSDF⁵⁵⁸ (LS) S_B20 ⁶⁵¹PIGAGICASYHT⁶⁶² (LS) S_B21 ⁶⁶³VSLLRSTSQKSI⁶⁷⁴ (LS) (RRAR inserted at this cleavage site) S_B22 ⁶⁹⁵AIPTNFSISITTEV⁷⁰⁸ (LS) M_B3 ¹⁶⁵KEITVATSRTLS¹⁷⁶ (100%) M_B4 ⁵GTITVEELKQLL¹⁶ (Q14K)	The structural (S, E, M, and N) proteins were synthesized as a series of 4942 10‐mer short overlapping peptide sequences. The peptides were tested for their potential recognition by neutralizing sera from four convalescent patients; healthy, unexposed patients; or by deceased patient sera. The Abs recognized by convalescent patient sera were potentially neutralizing and therapeutic; however, only simple linear epitopes can be recognized by this method. Other S‐, E‐, and N‐protein sequences were also found, but their protective efficacy is doubtful since they were either not found in three or more of the four sera (immune‐subdominant), or were not directed against M‐ or S‐proteins, as the other proteins are not as exposed on the virus surface. About 90% of produced Abs in convalescent SARS patients target the S‐proteins.		^[ ²¹² ^]
S_B22 ⁴⁶⁰FSPDGKPATPPALNAYW⁴⁷⁶ (neutralizing) S_B23 ⁶⁰⁹AIHADQLTPAWR⁶²⁸ (nonneutralizing) S_B24 ⁴⁶⁰FSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQ⁴⁹²	High‐affinity neutralizing & nonneutralizing Abs were produced by hybridoma cell fusion to the antigen. The neutralizing Abs in general were found to bind to the RBD region. The most potent Ab in this library (G18) was found to bind to linear S_B22 epitope and RBD with high affinity (1.78 nm), while nonneutralizing G8 was found to bind to a distant RBD epitope, S_B23, with very high affinity (0.83 nm). Other highly potent nAbs were conformational and bound to the same region rich in critical binding residues, e.g., nAbs 80R and m396 bind to the 469–492 region of the RBD of S_B24 with high binding affinities of 1.59 nm and 4.6 pm, respectively.^[ ¹⁴⁸, ²¹³ ^]		^[ ²¹⁴ ^]
S_T(h)25 ⁴³⁵NYNYKYRYLRGKLRPF⁴⁵¹ S_T(h)26 ⁵²⁸NFNGLTGTGVLTPSSKRF⁵⁴⁵ S_T(h)27 ⁶³³AGCLIGAEHVDTSYECDI⁶⁵⁰ S_T(h)28 ⁶⁴⁹DIPIGAGICASYHTVSLL⁶⁶⁶ S_T(h)29 ¹⁰⁸²SWFITQRNFFSPQII¹⁰⁹⁷ Orf_3T(h)1 ³⁶PLQASLPFGWLVIGV⁵⁰ M_T(h)5 ¹⁴⁶GHLRMAGHSLGRCDI¹⁶⁰ N_T(h)5 ²¹¹GETALALLLLDRLNQ²⁵⁵	The entire proteome of SARS‐1 was mapped for T‐cell epitopes by Elispot‐IFN‐g. Half of convalescent patients (n = 128) developed T‐cell responses. S‐protein was immunodominant as it covered most patients’ positive responses. 94% of recovered patients developed IgG titers against SARS‐1, and 91% of nAbs were directed against SARS‐1‐RBD with titers exceeding the 90% inhibitory concentration. This provides support for the theory that humoral responses are the more protective responses and correlates with infection survival estimates. nAb titers also correlated with T‐cell responses; however, T‐cell responses were not a prerequisite for recovery and seemed to develop slowly, which could be attributed to the inhibition of type‐1 INFs. In fatal SARS‐1 infection, serum levels of IL‐8, IL‐4, and IL‐5 were significantly higher than recovered patients’ levels (50, 3.4, and 0.64 pg mL⁻¹, respectively). S_T(h)25 epitope was highly recognized by T‐cells (22 of 64 patients), followed by S_T(h)27 (17 patients), and S_T(h)26 , S_T(h)28 , and S_T(h)29 (7 patients). The ORF3 sequence was the only peptide recognized by T‐cells more than S‐protein peptides (24/64). S_T(h)25 , as well as ORF3, were CD8⁺ epitopes. IL‐12 (20‐fold), IFN‐γ (10‐fold), IL‐10 (10‐fold), IL‐6 (30‐fold), and IL‐8 (3‐fold) were elevated compared to normal levels: 3, 3, 1, 2, and 30 pg/mL, respectively, in serum seven days post‐onset.		^[ ¹⁸⁹ ^]
M_B6 ¹MADNGTITVEELKQLLEQWNLVI²³ M_B7 ¹³²LMESELVIGAVIIRGHLRMAGHPLGRCDIK¹⁶⁰	Convalescent sera from 40 patients were mapped for immunodominant epitopes against SARS‐1 M‐protein. C‐terminal epitope (M_B7) was more dominant and had higher titers against it in the sera. The most reactive epitopes of M_B6–7 are listed. There are three transmembrane regions in the M‐protein sequence, starting from residue 15 and ending with 99. Therefore, Ab reactivity increased with the exposed portion of the protein. No in vitro neutralization or challenge studies were conducted.		^[ ²¹⁵ ^]
SARS‐2 mapped epitopes
SARS‐2‐S B‐cell epitopes [25 ug dose] S2_B1 ³³⁰PNITNLCPFGEVFNATRFAS³⁴⁹ S2_B2 ³⁷⁰STFKCYGVSPTKLNDLCFTN³⁹⁵ S2_B3 ⁴⁵⁰NYLYRLFRKSNLKPFERDIS⁴⁶⁹ S2_B4 ⁴⁸⁰CNGVEGFNCYFPLQSTGFQP⁴⁹⁹ SARS‐2‐S T‐cell epitopes S2_T(h)5 ⁴²⁰DYNYKLPDDFTGCVIAWNS⁴³⁹ S2_T(h)6 ⁵¹⁰RVVVLSFELLHAP⁵²¹	BALB/c mice (n = 5) S2_B2, S2_B3, or S2‐RBD were conjugated to KLH and mixed with alum SC (D 0, 14)	Epitopes from SARS‐2 N‐ and S‐proteins were mapped using 39 convalescent patients’ sera. Nine S‐protein immunodominant epitopes were identified (four epitopes in the RBD), compared to healthy volunteer sera. Two epitopes (S2B2, S2B3) were synthesized and tested as vaccine antigen in mice. They produced nAbs that had neutralizing activity comparable to the anti‐RBD Abs used as positive control. S2B2 and S2B3 contain also more likely T‐cell epitopes as shown by Elispot analysis, but S2_T(h)5 and S2_T(h)6 (S2_T(h)6 > S2_T(h)5) were potent in inducing IFN‐γ production from T‐cells.	^[¹⁰³, ²⁰⁴^]
SARS‐2 CD4⁺ T‐cell epitopes N2_T(h)1 ⁸¹DDQIGYYRRATRRIR ⁹⁵ N2_T(h)2 ²⁶⁶KAYNVTQAFGRRGPE²⁸⁰ N2_T(hc)3 ³²¹ GMEVTPSGTWLTYIGAIKLD³⁴⁰ SARS‐2 CD8⁺ T‐cell Within N2T(hc)3 MEVTPSGTWL	COVID‐19 convalescent patients (n = 37), Unexposed patients (n = 37), and SARS‐patients (n = 24)	Le Bert et al. explored virus‐specific T‐cell immunity against SARS‐2 epitopes from ORF‐1‐derived NSPs (esp. NSP‐7 and ‐13) and N‐protein via T‐cell Elispot. The specific T‐cell responses against ORF‐1 NSPs did not differentiate between unexposed patients and COVID‐19 recovered patients. In contrast, N‐protein, which is highly similar to SARS‐1‐N, matured T‐cells into CD4⁺, via N2_{T (h)1} (19% maturation) and N2_T(h)2 (2% maturation), and CD8⁺ T‐cells, via N2T₃ (2% maturation of the total population tested). N‐protein epitopes should, therefore, be included in peptide vaccines.	^[ ¹³⁴ ^]
S2_B5 ⁶⁵⁵HVNNSYECDIPIGAGICA⁶⁷² S2_B6 ⁷⁸⁷QIYKTPPIKDFGGFNFSQILPDPSKPSKPSKRSFIEDLL⁸²²	COVID‐19 convalescent patients (n = 12)	Linear neutralizing epitopes were mapped in SARS‐2 convalescent patients’ plasma. The epitopes are located at cleavage sites S2B5 at S1/S2, and S2B6 at S2’. The inhibition capacity of recovered patient Abs against S‐protein was tested via co‐incubation and gel electrophoresis.	^[ ²¹⁶ ^]
B‐cell epitopes S2_B7 ⁴⁵¹YLYRLFRKSNLKPFERDIST⁴⁷⁰ S2_B8 ⁴⁹¹PLQSYGFQPTNGVGYQPYRV⁵¹⁰ S2_B9 ³⁹¹CFTNVYADSFVIRGDEVRQI⁴¹⁰ S2_B10 ³³¹NITNLCPFGEVFNATRFASV³⁵⁰ S2_B11 ³⁴¹FNATRFASVYAWNRKRISN³⁶⁰ S2_B12 ⁴⁴¹LDSKVGGNYNYLYRLFRKSN⁴⁶⁰	COVID‐19 convalescent patients sera (n = 7) BALB/c Mice (n = 4) Cysteinylated peptides were conjugated to diphtheria toxoid (DT) and 25 µg were mixed with Alum IM (D 0, 21, 28)	Seven RBD‐derived epitopes were mapped in convalescent patients’ sera by screening 20‐mer overlapping peptides. The peptides were conjugated to diphtheria toxoid (DT). DT‐peptides immune sera were tested for ACE‐2/RBD binding inhibition using competitive ELISA technique. While these sera triggered modest inhibition <30%, at 1/4 serum dilution. However, combining S2_B7 and S2_B8 immune sera resulted in significant inhibition ranging from 75% at 1/4 serum dilution down to an inhibition of 55% at 1/32 serum dilution. The combination of S2_B7 and S2_B9 was less effective than above. The peptides were not the most immunodominant epitopes in convalescent patient sera among RBD‐derived epitopes.	^[ ²¹⁷ ^]
N2_T4 RTATKAYVN N2_T5 IIWVATEGA N2_T6 NTASWFTALT S2_T7 SIIAYTMSL S2_T8 ²⁶⁹YLQPRTFLL²⁷⁷ S2_T9 RVVVLSFEL	N‐protein CD8⁺ T‐cell epitopes were identified in three convalescent patients’ PBMCs with different HLA‐I A alleles: A02:06, A24:02, A02:01, A 03:01, and A24:07. Allele A02:01 patient, which is one of the most common MHC‐I alleles, recognized N2T₅. Other S‐ and N‐proteins epitopes were derived computationally with high allele‐population coverage, e.g., N2T₆, S2T_7‐9. S2T₈ was confirmed in separate studies by flow cytometry in convalescent patients PBMCs.^[ ²¹⁸ ^]		^[ ²⁰¹ ^]
Within S2_T8 ²⁶⁹YLQPRTFLL²⁷⁷ S2_T10 ⁹⁷⁶VLNDILSRL⁹⁸⁴ Orf₁2_T1 ³¹⁸³FLLNKEMYL³¹⁹¹	S‐protein derived A*02:01 allele restricted CD8⁺ T‐cell epitopes were identified in convalescent patient PBMCs, thus, priming host with these epitopes could help develop protective immune responses against infection. S2T₈, S2T₁₀, and Orf₁2T₁ stimulated IFN‐γ proliferation in PBMCs.		^[ ²¹⁸ ^]
Orf₁2_T2 TTDPSFLGRY Orf₁2_T3 VYIGDPAQL Orf₈2_T1 SKWYIRVGARKSAPL S2_T11 LTDEMIAQY S2_T12 QYIKWPWYI S2_T(h)13 ITRFQTLLALHRSYL M2_T1 NRFLYIIKL M2_T(h)2 LSYYKLGASQRVAGD N2_T7 KTFPPTEPKK N2_T8 ATEGALNTPK N2_T9 MEVTPSGTWL N2_T(h)13 KDGIIWVATEGALNT N2_T(h)14 GTWLTYTGAIKLDDK N2_T(h)15 RWYFYYLGTGPEAGL	PBMCs from 180 convalescent patients’ vs uninfected controls were used to examine CD4⁺ and CD8⁺ T‐cell epitopes throughout all of SARS‐CoV‐2 ORFs. The examination spanned common ten HLA‐1 alleles and six common HLA‐2 DR alleles. HLA‐1 alleles in CD8⁺ T‐cells A01 (80%), A01 (50%), A03 (64%), A11 (82%), A24 (70%), B40 (75%) and C*07 (55%) were stimulated (%) for IFN‐γ production via epitopes Orf₁2T_2, S2T₁₁, N2T_7, N2T_8, Orf₁2T_3, N2T_9, and M2T1, respectively. Similarly, HLA‐2 DR alleles in CD4⁺ T‐cells were highly stimulated 91%, 77%,73%, 55%, 95%, and 68%, by N2T_(h)13, N2T_(h)14, N2T_(h)15, S2T_(h)13, M2T_(h)2 epitopes, respectively. Several of these epitopes were found to stimulate nonexposed PBMC T–cells in milder manner compared to convalescent PBMC samples, e.g., N2T_(h)13 (44%) due to sequence similarity with common cold corona viruses. In terms of T‐cell stimulation by structural and nonstructural whole proteins, HLA‐1 overall stimulation was in the following rank order S‐protein > M‐protein > Orf3, while HLA‐2 DR overall stimulation rank order was M‐protein >ORF8> E‐protein>N‐protein.		^[ ²¹⁹ ^]
Orf₁2_T(h)4 ³³²⁶NHNFLVQAGNVQLRV Orf₁2_T(h)5 ⁵³¹SPLYAFASEAARVVR S2_T(h)14 ⁸¹⁶SFIEDLLFNKVTLAD S2_T(h)15 ²³⁶TRFQTLLALHRSYLT S2_T(h)16 ³²¹QPTESIVRFPNITNL S2_T(h)17 ³¹⁶SNFRVQPTESIVRFP	CD4+ T‐cell epitopes were screened in convalescent patients (n = 20) and in uninfected PBMCs samples. Four potent S‐protein derived CD4⁺ T‐cell epitopes were identified, that induced very high stimulation in several DRB and DQA HLA‐2 alleles, two of which were located in RBD sequence (S2T_(h)16–17), while two others (S2T_(h)14–15) were more potent and were located outside of RBD. Two more epitopes from Orf1 were also very potent in helper T‐cell stimulation. Overall helper T‐cell stimulation was higher for S‐protein than ORF1.		^[ ²²⁰ ^]
Within S2_T8 ²⁶⁹YLQPRTFLL²⁷⁷ S2_T18 ¹⁰⁰⁰RLQSLQTYV¹⁰⁰⁸	Convalescent patients (n = 17) and unexposed PBMCs were tested for CD8⁺ T‐cell stimulation with HLA‐1 A*02:01 restricted epitopes from SARS‐2‐S. S2T₈ and S2T₁₈ stimulated INF‐γ from CD8⁺ T‐cells of most patients’ PBMCs.		^[ ²⁰² ^]
BALB/c Epitopes N2_T(h)16 ³⁵¹ ILLNKHIDAYKTFPP³⁶⁵ S2_T19 ²⁶⁸GYLQPRTF²⁷⁵ S2_T20 ⁵³⁵KNKCVNFNF⁵⁴³ C57BL/6 Epitopes Orf₃2_T(h) ²⁶⁶EPIYDEPTTTTSVPL²⁸⁰ M2_T3 ¹⁷⁴RTLSYYKL¹⁸¹ N2_T17 ²¹⁹GFSALEPL²²⁶ S2_T21 ⁵¹⁰VVVLSFEL⁵¹⁷ S2_T22 ⁵³⁸CVNFNFNGL⁵⁴⁶ S2_T23 ⁸²⁰DLLFNKVTL⁸²⁸	Epitopes were initially mapped in hACE2‐transgenic BALB/c and C57BL/6 mice to identify potent CD4⁺ T‐cell and CD8⁺ T‐cell epitopes and to examine their protective efficacy. A nanomolar dose of each peptide was adequate to stimulate IFN‐γ production in T‐cells within mice lungs and bronchoalveolar fluid. S2_T22 and S2_T23 were the most potent epitopes in hACE2‐C57BL/6 mice, while S2_T20 was the most potent for hACE2‐BALB/c. Immunization of mice (hACE2‐BALB/c or hACE2‐C57BL/6) with N2_T(h)16, S2_T19, or S2_T20 resulted in only partial protection in challenge experiments: 5‐ to 10‐fold PFU reductions in SARS‐2 virus were observed on the second day post‐challenge.		^[ ²⁰³ ^]
S2_B13 ⁶³TWFHAIHVSGTNGTKRFDNPV‐LP⁸⁵ S2_B14 ⁹²FASTEKSNIIRGWIF¹⁰⁶ S2_B15 ¹³⁹F LGVYYHKNNKSWM¹⁵³ S2_B16 ⁴⁰⁶EVRQIAPGQTGKIAD⁴²⁰ S2_B17 ⁴³⁹NLDSKVGGNYNYLYR⁴⁵⁴ S2_B18 ⁴⁵⁵FRKSNLKPFERDIS⁴⁶⁹ S2_B19 ⁴⁷⁵GSTPCNGVEGFNCYFPLQSYGF‐QP⁴⁹⁹ S2_B20 ⁴⁹⁵GFQPTNGVGYQPYR⁵⁰⁹ S2_B21 ⁷⁹³PIKDFGGFNFSQILPDPSKP⁸¹² S2_B22 ⁹⁰⁹GVTQNVLYENQKLI⁹²³ S2_B23 ¹¹⁰⁶QRNFYEPQIITTDNT¹¹²⁰	Thirty‐three potential epitopes from various SARS‐2 proteins were used to immunize BALB/c mice and generate Ab titers. The generated Abs were evaluated for their neutralization efficacy using pseudovirion neutralization assays against the original SARS‐2 strain and the D614G mutant. The S2_B14 and S2_B15 epitopes neutralized both strains, while other epitopes were only effective against the D614 strain (S2_B13). Antisera from NTD‐derived epitopes (S2_B13‐S2_B15) were highly neutralizing. Epitopes that were less protective against the D614 strain (S2_B19‐S2_B29) generated nAb titers of ≤ 30; moderately protective epitopes against both original and D614G strains (e.g., S2_B15 and S2_B19), and against the D614 strain (S2_B14, and S2_B18) generated nAb titers of 30–50; and epitopes that were highly protective against the G614 strain (S2_B13, S2_{B 14,} S2_B21‐S2_B23) generated nAb titers of ≥ 50. CTD‐ and NTD‐derived epitopes (S2_B13, S2_B23) were more effective against D614G; however, RBD‐derived epitopes (S2_B16, S2_B17) were only slightly less effective. Notably, longer epitopes tend to adopt a conformation similar to those of the native protein, resulting in more potent nAbs.		^[ ²²¹ ^]

^{^a)}

S_T1 ¹¹⁷⁴NLNESLIDL¹¹⁸² is SARS‐1‐S derived T‐cell epitope, at position 1174 to 1182 of the full‐length Spike protein sequence. N and M, denote SARS‐1‐N and SARS‐1‐M proteins derived epitopes, while S2 denotes SARS‐2‐S derived epitopes, N2 and M2 denote SARS‐2‐N and SARS‐2‐M proteins derived epitopes, respectively. The B subscript denotes a B‐cell epitope, T subscript denotes T‐cell epitope, and T(h) subscript denotes a helper T‐cell epitope. SARS‐1‐derived epitopes with low sequence identity to SARS‐2 (LS) have more than 3 different residues.

^{^b)}

(IN), C (42) is challenge on day 42 from day zero (immunization), infection via intranasal route.

Ab, antibodies; C, challenge; D, day; DC, dendritic cells; HLA, human leukocyte antigen system; IM, intermuscular; IN, internasal; IP, intraperitoneal; KLH, Keyhole limpet hemocyanin; LPS, bacterial lipopolysaccharide, pro‐inflammatory Th‐2 adjuvant; MA15, a mouse adapted SARS‐1 strain; MHC, major histocompatibility complex; nAb, neutralizing antibody; PFU, plaque forming unit; SC, sub‐cutaneous; TCID, tissue culture infective dose 50%.