HCoV-NL63) and beta-CoVs (HCoV-OC43 and HCoV-HKU1) are well adapted to 68 humans and widely circulate in the human population, with most infections causing 69 mild disease in immunocompetent adults (3, 5, 6). In addition, SARS-CoV, 70 SARS-CoV-2 and MERS-CoV belong to Beta-CoV and are highly pathogenic (7-9). As the primary glycoprotein on the surface of the viral envelope, the spike (S) 80 glycoprotein is the major target of neutralizing antibodies (nAbs) elicited by natural 81 infection and key antigens in experimental vaccine candidates. The S protein contains 82 two subunits responsible for receptor binding (S1 subunit) and membrane fusion (S2 83 subunit) (11). In particular, the S1 subunit of the prefusion S protein is structurally (17, 18) . The S1 subunits of Beta-and Gamma-CoV strains utilize the cross-subunit 103 packing mode, reducing the conformational conflict of the RBD in a standing state 104 (13, 19, 20, 24). In contrast, Alpha-and Delta-CoV strains both utilize an intrasubunit 105 packing mode, and the S1-CTD is limited by the conformational conflict with 106 surrounding domains (12, 14, 16-18, 21, 24) . Hence, the S1-RBD in the S trimer was 107 captured in two different states among different coronaviruses. In the Beta-CoVs 108 (SARS-CoV, SARS-CoV-2 and MERS-CoV), the S1-RBD adopts a "standing" state, 109 which is believed to be a prerequisite for receptor binding and RBM-specific antibody 110 6 binding (13, 19, 20) . Nevertheless, the S1-RBDs of alpha-CoVs all adopt "lying" state, 111 which is considered more conducive to evading antibody recognition (12, 14, 16, 21 MERS-CoV. Among them, the S protein or RBD was the major targets (45) (46) (47) .
Compared with Beta-CoVs, relatively few studies have investigated two 123 alpha-hCoVs: HCoV-229E and HCoV-NL63. However, their S1 subunit structure and 124 receptor recognition pattern, especially the structure of the RBD and its state in the S 125 trimer, differ substantially from those of beta-CoVs, suggesting different S protein 126 immune responses between alpha-and beta-CoVs. Importantly, considering the low 127 homology between different coronavirus genera, related research on alpha-CoVs can 128 not only help to elucidate the differences between S proteins that adopt different RBD 129 states but can also facilitate the development of coronavirus vaccines. In this study, 130 we selected SARS-CoV, SARS-CoV-2, and HCoV-229E as models, which adopt the 131 two RBD states, and evaluated and compared immune responses to the S trimers and 132 7 RBDs of these coronaviruses through immunological and bioinformatics approaches. 133 We also investigated the mechanism through which the HCoV-229E S trimer 134 produced effective nAbs. Finally, we provide possible vaccine strategies for alpha- To address this issue, we performed B-cell epitope predictions for the S trimers 152 and RBDs of alpha-CoV (HCoV-229E) and beta-CoVs (SARS-CoV and 153 SARS-CoV-2). The predicted positive residues (the corresponding spatial epitope and 154 8 linear epitope) are displayed on the structural surface ( Fig. 2A, 2C and 2E) , and the 155 distribution of positive residues on the RBD is summarized in Table 1 . A total of 51 156 and 26 amino acid residues located on the RBD were predicted to be conformational 157 epitopes for SARS-CoV and SARS-CoV-2, respectively. Of these, 47 and 25 residues 158 were located in the SARS-CoV RBM subdomain and in the SARS-CoV-2 RBM 159 subdomain, respectively. The linear B-cell epitope prediction results were similar in 160 SARS-CoV and SARS-CoV-2. However, in HCoV-229E, only 3 residues located in 161 the RBM subdomain were predicted to be conformational epitopes, and 9 residues 162 were predicted to be linear epitopes. The same results also appeared in the 163 HCoV-229E S trimer: fewer positive residues were located in the RBD than in the 164 SARS-CoV or SARS-CoV-2 RBM subdomain ( Fig. 2A, 2C and SARS-CoV-2-immunized mice had a good neutralizing ability ( Fig. 3I and 3J) . 205 For HCoV-229E, the S trimer serum had a comparable neutralizing ability to that of 206 SARS-CoV or SARS-CoV-2, but the RBD serum had no detectable neutralizing 207 ability (Fig. 3K) . Our experimental results indicate that the lying state of the RBD in 208 the HCoV-229E S-trimer induces the production of very few antibodies targeting the 209 RBD, but the S-trimer still produces strong neutralizing antibody levels.
In this study, we found that more RBD-specific antibodies were induced by the 211 S trimer with the RBD in the standing state than the S trimer with the RBD in the 212 lying state, and the affinity between RBD-specific antibodies and the S trimer was 213 also higher in the standing state. However, we also found that fewer nAbs were 214 induced by the RBD of HCoV-229E than by the RBDs of SARS-CoV or 215 SARS-CoV-2. In terms of HCoV-229E, the distribution of the potential residues in the 216 RBM was lower than that of SARS-CoV or SARS-CoV-2, which may have been 217 caused by different RBM patterns and exposure degrees. When we compared the 218 reported nAb epitopes of SARS-CoV and Alpha-CoV TGEV with our results (47), 219 they were basically consistent. Therefore, we believe that this finding illustrates the 220 11 inherent difference between the RBDs of alpha-and beta-CoV. 221 The intact and stable S1 subunit of HCoV-229E is a prerequisite for the 222 production of effective nAbs 223 Our experimental results showed that HCoV-229E S-trimer can induce strong 224 nAb levels, while the RBD alone is less immunogenic. Next, we will explore which 225 functional domains of the S-trimer are involved in the generation of nAbs. To clarify 226 this issue, we immunized mice with the HCoV-229E S trimer (10 µg), S1 (10 µg), 227 NTD (10 µg), RBD (10 µg) and NTD+RBD (5 µg+ 5 µg). Meanwhile, to better 228 confirm our results, the HCoV-229E strain VR740 was used for the neutralizing assay. 229 The results indicated that the S trimer serum had the best neutralizing ability, followed 230 by the S1 and NTD+RBD sera, while the NTD and RBD sera alone had no detectable 231 neutralizing effects (Fig. 4A) . The results indicate that the S1 region in the S-trimer 232 should be the key region for nAbs induction. To further verify the importance of the 233 complete S1 structure in the S-trimer, we designed two S trimer mutants, namely, an 234 NTD-deficient S trimer and an S65C/T472C S trimer, the S1 subunit integrity or 235 stability of which was destroyed ( Fig. 4C and 4F ). Mutant proteins disrupt the 236 conformational conflicts that limit RBD standing, significantly improving their ability 237 to bind hAPN ( Fig. 4D and 4G) . However, an incomplete or unstable S1 238 conformation significantly reduces the level of nAbs induced by the S-trimer (Fig. 4E   239 and 4K). Taken together, these results showed that the intact and stable S1 subunit of 240 HCoV-229E is a prerequisite for the production of effective nAbs.
Furthermore, our experimental results show that RBD has a higher ability to bind 242 12 to the receptor hAPN (Fig. 4B) , which indicates that the characteristics of RBD itself 243 may lead to the generation of less neutralizing antibodies. Furthermore, we screened 244 monoclonal antibodies using S-trimer, and the results showed that few antibodies 245 targeting S1-RBD (Fig. 5A) . To further determine the ability of RBD to induce 246 antibodies itself, we screened monoclonal antibodies targeting the S1 region and 247 found that the proportion of antibodies targeting RBD was approximately 20% (Fig.   248   5B ). Since the S1 protein is expressed in a monomeric form, RBD is not restricted by 257 We compared the structures of S trimers and RBDs among alpha-coronaviruses 258 (Figs. 1B and 6A) . We also predicted the potential B-cell epitopes for their RBDs 259 ( Fig. 6A; Table1) . In Alpha-CoV, the S-trimer had a closed S1 subunit with three 260 "lying" RBDs (Fig. 1B) . Moreover, the RBDs consist of a standard β-sandwich fold 261 core and three short discontinuous loops in the same spatial region (12, 14, 16, 21, 26, 262 27, 48) (Fig. 6A) . Meanwhile, we performed a structural conservative analysis and the 263 results showed that the RBD structures of HCoV-NL63, PEDV, and FIPV are most 264 13 similar to HCoV-229E, with RSMD values of 1.9, 2.0, and 2.2, respectively (Fig. 6B) . 265 In addition, the distribution of potential B-cell epitopes in the RBDs of alpha-CoVs 266 was also similar to that of HCoV-229E (Fig. 6A and 6C; Table1) . Based on the above 267 data, inherent differences exist in the RBDs between alpha-and beta-CoVs (Figs. 2   268 and 6A). However, the alpha-and beta-CoVs show high similarity in their RBDs and 269 similar potential immune characteristics within their respective genera (Figs. 2, 3, 6A   270 and 6B). Accordingly, in alpha-CoVs such as HCoV-229E, subunit vaccines should 271 prioritize the S-trimer rather than the RBD. In beta-CoVs such as SARS-CoV and 272 SARS-CoV-2, the S trimer and RBD are both good candidates for subunit vaccines 273 (Fig. 7) . 274 In summary, we systematically analyzed the conformational states and IgG (1:5,000 diluted in PBST with 1% BSA (w/v), Boster) was used for detection.
Signal reading was carried out in the same manner. HBS buffer was used as a mock 
Then the plates were reacted with the hybridoma culture supernatants at 37℃ for 1h.
HRP-conjugated goat anti-mouse IgG (1:5,000 diluted in PBST with 1% BSA (w/v),
Boster) was used for detection. Signal reading was carried out in the manner 368 described above. Hybridoma culturing medium was used as a mock control. 