key: cord-0900865-em1yuym2
authors: Guo, Chang; Peng, Yanan; Lin, Lin; Pan, Xiaoyan; Fang, Mengqi; Zhao, Yun; Bao, Keyan; Li, Runhan; Han, Jianbao; Chen, Jiaorong; Song, Tian-Zhang; Feng, Xiao-Li; Zhou, Yahong; Zhao, Gan; Zhang, Leike; Zheng, Yongtang; Zhu, Ping; Hang, Haiying; Zhang, Linqi; Hua, Zhaolin; Deng, Hongyu; Hou, Baidong
title: A pathogen-like antigen based vaccine confers immune protection against SARS-CoV-2 in non-human primates
date: 2021-10-23
journal: Cell Rep Med
DOI: 10.1016/j.xcrm.2021.100448
sha: 033ab75f54426aad6621e458d47d3f15a8ab5c6e
doc_id: 900865
cord_uid: em1yuym2

Activation of nucleic acid sensing Toll-like receptors (TLRs) in B cells is involved in antiviral responses by promoting B cell activation and germinal center responses. In order to take the advantage of this natural pathway for vaccine development, synthetic pathogen-like antigens (PLA) constructed of multivalent antigens with encapsulated TLR ligands can be used to to activate B cell antigen receptors and TLRs in a synergistic manner. Here we report a PLA-based COVID-19 vaccine candidate designed by combining a phage-derived virus-like particle carrying bacterial RNA as TLR ligands with the receptor-binding domain of SARS-CoV-2 S protein as the target antigen. This PLA-based vaccine candidate induced robust neutralizing antibodies in both mice and non-human primates (NHPs). Using a NHP infection model we demonstrated that the viral clearance was accelerated in vaccinated animals. In addition, the PLA-based vaccine induced Th1 oriented response and a durable memory, supporting its potential for further clinic development.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a new virus causing coronavirus disease 2019 , has caused more than 4 million deaths after 18 months of 48 its emergence (World Health Organization (WHO)). The pandemic imposed enormous burdens 49 on medical care, economy and social life. Several types of vaccines have been approved for 50 clinical use worldwide, including inactivated virus, non-replicating viral vector and mRNA-51 based vaccines 1 . Although most countries are actively promoting the vaccination process, new 52 waves of infections with different viral variants continue to be the major concern for the public 53 health 2 . Reluctance to vaccination is one of the major problems for achieving the herd 54 immunity, and concerns for the safety and side effects of vaccination have always been an issue. 55

In addition, the long-term efficacy and potential serious side effects for the current approved 56 COVID-19 vaccines are still under examination. It is therefore worthy of continuing to develop 57 other types of COVID-19 vaccines with less concern of safety and a more durable effect. Indeed, 58 in addition to the above-mentioned three types of vaccines, DNA, protein subunit and virus-like 59 particle (VLP) based vaccine candidates are also under clinical or pre-clinical development 60 worldwide (https://www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-61 vaccines). 62

Although there have been many successful vaccines in human history such as those for smallpox, 63 polio and measles, most of these prophylactic vaccines were developed by a trial-and-error 64 approach. Not all the infectious diseases can be prevented by vaccines despite the advances in 65 both basic research and biopharmaceutical technology. An incomplete understanding of how our 66 immune system responds to different types of infections as well as to those successful vaccines 67 has hindered our progress in vaccine development. It is generally accepted that antigen-68 presenting cells (APCs) especially dendritic cells (DCs) are important in the initial activation of 69 the adaptive immune responses 3 . However, we recently found that B cells instead of dendritic 70 cells (DCs) could serve as the dominant APCs to activate CD4 + T cells upon immunization with 71 phage Qβ-derived VLPs (Qβ-VLP) or inactivated influenza virus 4 , suggesting that alternative 72

APCs could be used to activatate the immune response. The nucleic acid sensing Toll-like 73 receptors (TLRs) in B cells is essential for their antigen presenting function as it activates B cells 74 to secrete factors that can promote CD4 + T cell differentiating towards Tfh and Th1 4 . Moreover, 75 B cell TLR signaling has been shown to be involved in anti-viral responses in multiple cases 76 J o u r n a l P r e -p r o o f 5 through promoting B cell proliferation and differentiation including germinal center (GC) 77 response [5] [6] [7] [8] [9] . Dependence on B cell TLR signaling for anti-viral reponses is likely an 78 evolutionarily conserved mechanism in both mice and humans. Human B cells express similar 79 endosomal nucleic acid-sensing TLRs as mice do 10 . In addition, the pathological role of TLRs in 80 systemic lupus erythematosus, an autoimmune disease characteristic with anti-nuclear antibody, 81 seems to be conserved between mice and humans, suggesting that the same pathway might be 82 reserved for the immune responses to infections 11, 12 . Interestingly, a recent study identified loss-83 of-function TLR7 variants being associated with severe COVID-19 in young male patients 13 , 84 supporting further that TLRs in humans are involved in the anti-viral immunity. 85

To take the advantage of this natural anti-viral mechanism in B cells for vaccine development, 86 antigens need to be presented in a multivalent form to maximize B cell antigen receptor (BCR) 87 activation as well as to carry the TLR ligands inside, so that the uptake of the TLR ligands could 88 be coupled to the BCR-mediated endocytosis to achieve a synergistic signaling effect in B cells 5, 89 [14] [15] [16] . To distinguish this type of VLPs from the other VLPs or nanoparticles that do not 90 encapsulate TLR ligands, we named them pathogen-like antigens (PLAs). In this study, we 91 conjugated the receptor-binding domain (RBD) of the SARS-CoV-2 S protein to a PLA platform 92 to build a COVID-19 vaccine candidate. We found that this PLA-based COVID-19 vaccine 93 candidate induced robust neutralizing antibodies, a Th1-oriented immune response, a long-94 lasting GC response, and the production of long-lived PCs and memory B cells in mice, all of 95 8 anti-viral response. From the aspect of B cells, cytokines associated with Th1 response tend to 168 promote Ig isotype switch to IgG2a/c in mice which is equivalent to IgG1 in humans, whereas 169

Th2 associated cytokines promote Ig isotype switch to IgG1 in mice which is equivalent to IgG4 170 in humans. Th1-associated IgG subclass is more potent in mediating antibody-dependent cellular 171 cytotoxicity and phagocytosis compared with Th2-associated IgG subclass, thus could contribute 172 to the anti-viral response more efficiently 30 . 173 We previously found that activation of TLR/MyD88 signaling in B cells by Qβ-VLP strongly 174 promoted the immune response towards Th1 direction, with the induction of T-bet in cognate 175 CD4 + T cells and Ig class-switch to IgG2a/c 4, 9 . To determine whether AP205-RBD induced 176 response is Th1-oriented, we first examined the Ig isotypes of anti-RBD antibodies. As expected, 177 AP205-RBD induced high titers of IgG2a/c ( Figure 3A ), and the ratio of IgG2a/c to IgG1 was 178 significantly higher in AP205-RBD immunized group compared with the soluble RBD 179 immunized groups ( Figure 3B ). Addition of CpG on top of alum could indeed increase the 180 IgG2a/c to IgG1 ratio in soluble RBD immunized groups as expected, but could not bring it to 181 the same level as that of the AP205-RBD immunized group ( Figure 3B) . 182

Since the absolute quantitiy of different Ig isotypes cannot be compared directly by ELISA, we 183 further quantified RBD-specific plasma cells (PCs) by ELISpot assay. There were significantly 184 more IgG2a/c + PCs than IgG1 + or IgG2b + PCs in both splenocytes and bone marrow (BM) cells 185 (Figures S3A and S3B) , consistent with the ratio of anti-RBD IgG2a/c to IgG1 determined by 186 ELISA ( Figure 3B ). The potential effect of LPS on AP205-RBD induced antibody response was 187 also examined. Using AP205-RBD with the LPS level differing by ~100,000 fold, we found no 188 significant difference in either the total anti-RBD IgG titer or the IgG subclass distribution from 189 the immunized mice ( Figure S3C ), suggesting that LPS did not contribute to the Th1-oriented 190 response induced by To further evaluate whether AP205-RBD induced Th1 response, IFNγ secreting cells were 192 examined in splenocytes from naïve or immunized mice. The 15-mer peptides derived from the 193 RBD sequence 31 or the intact RBD protein were used for in vitro stimulation. There were a 194 number of cells secreting IFNγ upon peptide stimulation in AP205-RBD immunized mice 195 whereas very few of these cells were found in naïve or mice immunized with soluble RBD plus 196 alum ( Figures 3C-D) , suggesting that AP205-RBD indeed induced Th1-oriented response. We 197 J o u r n a l P r e -p r o o f 9 cannot distinguish if these IFNγ secreting cells were CD4 + or CD8 + T cells, although we found 198 previously that similar kind of VLPs induced robust T-bet expression in CD4 + T cells 4 and a 199 rather moderate CD8 + T cell response (unpublished) . Overall, the IgG2a/c dominated antibody 200 response and the generation of abundant IFNγ secreting cells supported that AP205-RBD 201 induced a Th1-oriented response. 202

The PLA-based COVID-19 vaccine candidate elicited antigen-specific germinal center 203 (GC) response. 204 GC response is a specialized form of T-dependent antibody response, in which GC B cells with 205 the help from Tfh cells undergo multiple rounds of cell divisions, and go through somatic 206 hypermutation for affinity maturation 32 . GC response is also involved in the generation of long-207 lived plasma cells and memory B cells. We previously found that Qβ-VLP could induce not only 208 a robust but also a prolonged GC response 33 , which depends on B cell TLR/MyD88 signaling 9 . 209

To test whether AP205-RBD could induce GC response in mice, antigen-specific B cells labeled 210 with fluorophore-conjugated antigens were examined by flow cytometry. Upon AP205-RBD 211 immunization, both RBD-specific and AP205-specific PCs, GC B cells and memory B cells with 212 switched isotype (swIg MemB) were detected, wherease naïve mice only contained IgD + /IgM + 213 naïve antigen + B cells, supporting that AP205-RBD induced antigen-specific GC response 214 ( Figure 4A ). Fluorophore-binding B cells maintained as IgD + /IgM + naïve B cells upon AP205-215 RBD immunization ( Figure S4A -C), supporting our staining strategy. The antigen + GC B cells 216 reached the peak level at about 2 weeks after the first immunization ( Figure 4B ), which is 217 consistent with what we have observed previously for the other PLAs 33 . Interestingly, a second 218 immunization did not further increase the GC B cell number ( Figure 4B ), suggesting that one 219 dose of AP205-RBD is enough to launch the full-scale GC response. Nevertheless, a second 220 immunization led to a boost of antigen + PCs ( Figure 4B ), which is consistent with the increased 221 antibody titers upon the second immunization and presumably would benefit the vaccinees upon 222 infection. Moreover, AP205-RBD induced GC response lasted quite long, with RBD-specific GC 223 B cells being detectable even 2 months after the second immunization ( Figures 4A-B) , which is 224 also consistent with the duration of the GC response elicited by a similar kind of PLA 33 . 225

Anatomical GC structures in spleens were also identified by immunohistochemistry in the 226 immunized mice (Figures 4C-D) . 227

The PLA-based COVID-19 vaccine candidate elicited durable humoral memory. 228

Humoral memory, the immunological memory formed by B cells, consists of long-lived PCs and 229 memory B cells. The long-lived PCs are responsible for maintaining antigen-specific antibody 230 level and are generated through GC response. Memory B cells could be highly heterogeneous, 231

with IgM + memory B cells usually formed during the early response but with low affinity to the 232 antigen, and the swIg memory B cells formed through the GC response with increased affinity 233 and other gene expression changes 34, 35 . This latter group of memory B cells are usually more 234 responsive and tend to differentiate into plasma cells more readily upon the reencountering of 235 antigen. 236

To evaluate AP205-RBD induced humoral memory, RBD-specific long-lived PCs were 237 examined in mice immunized 3 to 4 months previously. Since long-lived PCs reside in BM 36 238 and can also be found in spleen as we previously showed 33 , both splenocytes and BM cells were 239 examined by ELISpot for RBD-specific IgG secreting cells. Indeed, RBD-specific PCs were 240 abundant in both spleen and BM even 4 months after immunization ( Figures 5A-B) . 241

Consistently, we found that the anti-RBD IgG maintained relatively stable from 2 month after 242 the second immunization up till a year as we have followed ( Figure 5C ). Besides long-lived PCs, 243

we also found that a large proportion of RBD-specific swIg memory B cells were IgG2a/c + 244 ( Figure S5A ). We previously showed that IgG2a/c + memory B cells induced by similar PLA 245 could last for more than a year in mice 33 . Therefore, AP205-RBD induced not only a robust 246 antibody response but also a durable humoral memory. 247

Since AP205-RBD induced both anti-AP205 and anti-RBD antibodies ( Figure S1C ), one concern 248 using AP205-RBD as a vaccine is whether the induced anti-AP205 antibody could reduce the 249 effect of other vaccines using the same AP205 as a carrier in the future. To address this question, 250

we immunized a group of mice that had received other AP205 based vaccine candidates 4-6 251 months ago with AP205-RBD. We found that there was no significant difference of the AP205-252 RBD-induced anti-RBD antibody levels between the mice with and those without a prior 253 immunization history ( Figure S5B ). On the other hand, the anti-AP205 antibody titers were 254 significantly higher in mice that have been immunized with another AP205 based antigens 255 previously as one might expect ( Figure S5B ). This result suggested that the pre-existing 256 J o u r n a l P r e -p r o o f 11 antibodies against the PLA carrier part did not affect significantly the antibody response towards 257 a new antigen target. 258

The PLA-based COVID-19 vaccine candidate elicited neutralizing antibodies in non-259 human primates. 260

To further evaluate the potential of AP205-RBD as a COVID-19 vaccine candidate for humans, 261 we immunized rhesus macaques i.m. with AP205-RBD twice 3 weeks apart ( Figure 6A ). Both 262 anti-RBD and anti-AP205 IgG were elicited after the first immunization and further increased 263 upon the second immunization ( Figure 6B ). In addition, sera collected from the immunized 264 macaques exhibited neutralizing activity against both the pseudovirus and live SARS-CoV-2 265 virus (Figures 6C-F). Curiously, it seems that sera from some unimmunized macaques already 266 exhibited a low level of neutralizing activity ( Figure 6F ). It is unclear whether these macaques 267 have been exposed to other coronaviruses previously as they were not raised in a strictly 268 pathogen-free environment. Nevertheless, the background neutralizing activity in the 269 unimmunized animals varied a lot whereas immunization with AP205-RBD significantly 270 increased the neutralizing activity against live SARS-CoV-2 virus, supporting the efficacy of 271 AP205-RBD as a candidate vaccine. Lung tissues taken at day 7 post challenge were further processed for histochemistry 301 examinations. Although the virus were cleared much more quickly in the immunized animals, 302 the pathological changes characteristic of interstitial pneumonia were still evident in these 303 animals, which was likely caused by the high dose of the viral infections initially. At low 304 magnification, lesions with a patchy or diffuse pattern were seen in both control and immunized 305 animals ( Figure 7F ). At higher magnifications, the pathological changes mainly manifested as 306 thickened alveolar septa and infiltration of lymphocytes ( Figure 7G ). Occasionally, cell debris 307 could be found in the air spaces ( Figure 7G , panel a), which might reflect the infection-related 308 cell damage. There was no infiltration of eosinophils or other granulocytes in all the tissue 309 sections examined from both the control and the immunized animals. In regions where cell 310 infiltration was severe, the air spaces almost completely disappeared ( Figure 7G , panel c). We 311 quantified the proportion of such kind of consolidated areas in each tissue section and found that 312 the immunized animals tended to have lower proportion of the consolidated areas than the 313 control animals ( Figure 7H ). We found no other significant differences of the pathological 314 changes between the control and the immunized groups. Overall, it seemed that immunization 315 with AP205-RBD could accelerate the viral clearance especially in lungs and guts, but may not 316 prevent the viral infection completely. 317 J o u r n a l P r e -p r o o f 13 Discussion 318

Here we presented evidence supporting targeting B cell TLR signaling, a physiological anti-viral 319 mechanism, as a valid strategy to develop a COVID-19 vaccine. The PLA structure enables the 320 coupling of the BCR signaling with the TLR signaling in B cells, which serves as a message to B 321 cells to initiate the anti-viral response. The activated B cells could then go through proliferation 322 and differentiation, and could also promote Tfh development which in turn fuels back the GC 323 response, all of which contribute to the anti-viral humoral response 16 . In this study, we found 324 that AP205-RBD induced (1) In this study we chose rhesus macaques as the animal model of SARS-CoV2 infection as they 390

probably represent most closely to the human infection 71 . All the rhesus macaques in this study 391 exhibited no apparent severe symptoms such as fever or dyspnea even after a relatively high dose 392 of viral challenge ( Figure S7 ), which is consistent with that the majority of human COVID-19 393 patients were not in severe situations 72 . The characteristic interstitial pneumonia histological 394 change in the macaques (Figure 7) is also consistent with the chest radiology changes in many 395 human patients 73 . We found in this study that although our COVID-19 vaccine could 396 undoubtedly accelerate the viral clearance it did not prevent the initial viral entry completely 397 ( Figure 7 ). This is also consistent with the current clinical data for other vaccines that the vaccine 398 protected against the severe disease more effectively than preventing the infection 74, 75 . The viral 399 load in the nasal cavity continued to be detectable at day 7 after infection even in the immunized 400 macaques ( Figure 7A IgG2a/c titer to IgG1 titer. One-way ANOVA test was used to determine the significant 480 difference among the 3 groups. Tukey's multiple comparisons test was then used to compare 481 between the AP205-RBD immunized group and one of the RBD protein immunized groups. The 482 adjusted p value was used to indicate the statistical significance ( ** < 0.01, *** <0.001). (C) 483

Representative data of IFNγ ELISpot assay. Splenocytes from naïve mice or mice immunized 484 with the second dose of AP205-RBD or RBD+Alum 5d previously were examined for IFNγ + 485 cells. 15-mer peptide pools derived from RBD sequence were used to stimulate the cells in vitro. 486

Pool1 and pool2 contain peptides derived from 420-459aa and 511-549aa of SARS-CoV-2 S 487 protein respectively. Intact RBD protein was also used for stimulation. for antigen-specific cells by RBD-BV650 or AP205-AF647 labeling. RBD-BV650 + and AP205-495 AF647 + cells are defined as antigen + cells for simplicity although they may consist a fraction of 496 fluorophore-specific cells which are IgD + IgM + (Figures S4A-B) . Antigen-specific plasma cells 497

(PC) (CD19 -B220 -IRF4 + ), germinal center ( 

Further information and requests for resources and reagents should be directed to and will be 552 fulfilled by the lead contact, Dr. Baidong Hou (baidong_hou@ibp.ac.cn) . 553

Requests for plasmids of PLA vaccine components and recombinant protein should be directed 555 to and will be fulfilled by the Lead Contact, Dr. Baidong Hou. All reagents will be made 556 available on request following completion of a Material Transfer Agreement. 557

 All data for this study are available without restriction from the Lead Contact upon 559 request. 560  This study did not generate code. 561  Any additional information required to reanalyze the data reported in this work paper is 562 available from the Lead Contact upon request. 563

All mice used in this study were housed under specific pathogen-free conditions, and their use 566 was approved by the Animal Care and Use Committee of the Institute of Biophysics, Chinese 567 Academy of Sciences. C57BL/6 mice were bred in-house or purchased from SPF Biotechnology 568

Co., Ltd (China). Roughly equal number of male and female mice of 8-16 weeks were used for 569 immunization. 

The SARS-CoV-2 strain 107 37 was obtained from the Guangdong Provincial CDC, Guangdong, 589

China. The SARS-CoV-2 virus (nCoV-2019BetaCoV/Wuhan/WIV04/2019) 28 was obtained 590 from the National Virus Resource. SARS-CoV-2 virus was passaged in Vero-E6 cells. 591

The DNA sequence encoding the major AP205 coat protein (Gene ID: 956335) was synthesized 595 and cloned into the pET21 vector. The sequence encoding SpyTag was fused at the C-terminus 596 of AP205 gene with a flexible linker to generate AP205-SpyTag. AP205-SpyTag encoded VLPs 597 were expressed and purified in a similar way as Qβ-VLP as described before 33 . Basically, E. coli 598 BL21 (DE3) transformed with pET21-AP205 or pET21-AP205-Spytag was grown in LB 599 medium and protein expression was induced with 0.1 mM IPTG (Yeasen biotech, China) at 37˚C 600 for 4-5 hours. VLPs were purified by CsCl density gradient centrifugation at 200,000 g for 22 601

hours. LPS was removed by repetitive extraction with Triton X-114. Endotoxin detection kits 602 (ToxinSensor Chromogenic LAL Endotoxin Assay Kit, GenScript, L00350C) was used to 603 determine the LPS level in purified AP205-Spytag VLPs according to the manufacturer's 604 J o u r n a l P r e -p r o o f 23 instructions. Purified VLPs were examined by SDS-PAGE with Commassie Blue staining, or 605 electrophoresis in 1% agarose gel followed by ethidium bromide staining. The assembly of VLPs 606 was confirmed by transmission electron microscopy. The protein concentration of the VLPs was 607 determined using Bradford assay. 608

The coding sequences for the receptor binding domain (RBD) corresponding to the 319aa -610 541aa of SARS-CoV-2 (YP_009724390) S protein and the full-length S protein were synthesized 611 and cloned into pCEP4 vector (Addgene). The sequence encoding the signal peptide from the S 612 protein and SpyCatcher was synthesized and fused at the N-terminus of the RBD, and a 12-mer 613 of histidine tag was fused at the C-terminus of the RBD to generate RBD-SpyCatcher. The 614 sequence encoding AviTag and a 12-mer of histidine tag was fused at the C-terminus of RBD to 615 generate RBD-AviTag. pCEP4-RBD, pCEP4-S, pCEP4-RBD-SpyCatcher or pCEP4-RBD-616

AviTag was transiently transfected into Expi293F cells using the PEI (Polyscience, 23966-1) 617

following the manufacturer's recommendations. Recombinant proteins were then collected from 618 the supernatants of the cell culture and purified by affinity chromatography using Ni-NTA 619 agarose (BBI Life Sciences, China). Purified proteins were examined by SDS-PAGE with 620

Commassie Blue staining, and the protein concentration was determined using Bradford assay. 621

To generate AP205-RBD, every 100μg of RBD-SpyCatcher was added to 400μg of AP205-622 SpyTag in PBS buffer and incubated on ice for more than 1 hour. 623

To detect antigen-specific cells, AP205-AF647 was generated by conjugating AF647 to the 625 purified AP205 VLPs using the AF647 labeling kit from Thermo Fisher Scientific as described 626 before 33 . Qb-AF647 33 was used to indicate potential AF647 + cells. 627

The DNA sequence encoding the BirA (Gene ID: 948469) was synthesized and cloned into the 628 pGEX vector (Addgene). E. coli BL21 (DE3) transformed with pGEX-BirA was grown in LB 629 medium and protein expression was induced with 0.1 mM IPTG at 18˚C for 18 hours. GST-BirA Immunization 638 10μg of AP205-RBD (dose indicates the mass of the RBD part) was injected once or twice 3 639 weeks apart intraperitoneally when not specified. For comparison of immunization routes, the 640 same doses of AP205-RBD were injected subcutaneously or intramuscularly. For comparison of 641 different antigens, 10μg of RBD or 50μg of full-length S protein mixed with alum, or with 642 additional 50μg of CpG-ODN was injected intraperitoneally. For immunization of AP205 and 643 RBD without conjugation, AP205 instead of AP205-SpyTag was mixed with RBD-SpyCatcher. 644

To determine the amount of antigen-specific immunoglobulins in serum, purified RBD without 646 SpyCatcher fusion region, or purified AP205-SpyTag was used to coat the plate. Serial diluted 647 sera were incubated with the plate, and the horseradish peroxidase (HRP)-conjugated anti-mouse 648

IgA, anti-mouse IgG (Bethyl Laboratories, USA), anti-mouse IgM, anti-mouse IgG1, anti-mouse 649

IgG2b, anti-mouse IgG2a/c, anti-mouse IgG3 (Southern Biotech, USA), or anti-monkey IgG 650 (Abcam, UK) was used for detection. The 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich) was 651 used as the HRP substrate, and the optical density at 450nm was measured by a microplate 652 reader (SpectraMax, Molecular Devices, USA). Antibody titers were determined as the 653 reciprocal of the highest dilution that gave an optical density value that was above ten times of 654 the standard deviation value measured from the serum-free wells. 655 

SARS-CoV-2 vaccines in development

SARS-CoV-2 Variants and Vaccines

Dendritic cells and the control of immunity

B Cells Are the Dominant Antigen-Presenting Cells that Activate 762

Cells upon Immunization with a Virus-Derived Nanoparticle Antigen

Selective utilization of Toll-like receptor and MyD88 signaling in B 766 cells for enhancement of the antiviral germinal center response

Toll-like receptor 7 controls the anti-retroviral germinal center response

Toll-like receptor 7 is required for effective adaptive 771 immune responses that prevent persistent virus infection

B Cell-intrinsic TLR7 signaling is required for optimal B cell 773 responses during chronic viral infection

B Cell-Intrinsic MyD88 Signaling Promotes Initial Cell Proliferation and 776

Differentiation To Enhance the Germinal Center Response to a Virus-like Particle

Toll-like receptors--sentries in the B-cell response

Integration of B cell 781 responses through Toll-like receptors and antigen receptors

Contribution of Toll-like receptor signaling to 784 germinal center antibody responses

Among Young Men With Severe COVID-19

Receptors, subcellular compartments and 790 the regulation of peripheral B cell responses: the illuminating state of anergy

Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like 794 receptors

The role of B cell antigen presentation in the initiation of CD4+ T cell 796 response

TLR9 signaling in 798 B cells determines class switch recombination to IgG2a

Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin

Bacterial superglue enables easy development of 807 efficient virus-like particle based vaccines

Convergent antibody responses to SARS-CoV-2 in convalescent individuals

Human neutralizing antibodies elicited by SARS-CoV-2 814 infection

Analysis of the SARS-CoV-2 spike protein 816 glycan shield reveals implications for immune recognition

SCB-2019), a protein subunit 819 vaccine candidate for COVID-19 in healthy adults: a phase 1, randomised, double-blind, 820 placebo-controlled trial

BNT162b 823 vaccines protect rhesus macaques from SARS-CoV-2

Safety 826 and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates

Safety and immunogenicity of a recombinant tandem-830 repeat dimeric RBD-based protein subunit vaccine (ZF2001) against COVID-19 in adults: two 831 randomised, double-blind, placebo-controlled, phase 1 and 2 trials

Fcgamma Receptor Function and the Design of Vaccination 839 Strategies

Immunogenicity of a DNA vaccine candidate for COVID-19

Germinal centers

Independent B Cell Responses to a Virus-like Particle

Memory B cells

Memory B Cells of Mice and Humans

Plasma cell development 850 and survival

Delayed severe cytokine storm and immune cell infiltration in SARS-853

CoV-2-infected aged Chinese rhesus macaques

Development of an inactivated vaccine candidate for SARS-CoV-2

A Universal Design of Betacoronavirus Vaccines against 859 COVID-19, MERS, and SARS

DNA 862 vaccine protection against SARS-CoV-2 in rhesus macaques

Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

Viral targets for vaccines against COVID-19

Single-874 shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques

A Thermostable mRNA Vaccine 881 against COVID-19

Phase 927 1 randomized trial of a plant-derived virus-like particle vaccine for COVID-19

A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike 932 protein receptor-binding domain induces potent neutralising antibody responses

Tars 935 K, Mohsen MO, Bachmann MF. AP205 VLPs Based on Dimerized Capsid Proteins 936

Accommodate RBM Domain of SARS-CoV-2 and Serve as an Attractive Vaccine Candidate

Vaccines (Basel)

Developing Covid-19 Vaccines at Pandemic 939

The role of cDC1s in vivo: CD8 T cell priming through cross-941 presentation

Reactogenicity Following Receipt of mRNA-Based 943 COVID-19 Vaccines

COVID-19 vaccine-associated immune 945 thrombosis and thrombocytopenia (VITT): Diagnostic and therapeutic recommendations for a 946 new syndrome

With mRNA COVID-19 Vaccines in Members of the US Military

Myocarditis Occurring After Immunization With 951 mRNA-Based COVID-19 Vaccines

Adenoviruses as vaccine vectors

Safety and efficacy of an 956 rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine: an interim analysis 957 of a randomised controlled phase 3 trial in Russia

Respiratory disease in rhesus macaques inoculated with SARS-CoV-961 2

Immune determinants of COVID-19 disease presentation and severity

Time Course of Lung Changes at Chest CT during Recovery from Coronavirus Disease 2019 966 (COVID-19)

Asymptomatic and Symptomatic SARS-CoV-2 Infections After BNT162b2 Vaccination in a 969 Routinely Screened Workforce

Single-dose administration and the influence of the timing of the booster dose on 973 immunogenicity and efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine: a pooled analysis of 974 four randomised trials

A vaccine targeting the RBD of the S protein of SARS-CoV-2 977 induces protective immunity

Development of an Inactivated Vaccine Candidate

with Potent Protection against SARS-CoV-2

Effect of Vaccination on 982

Household Transmission of SARS-CoV-2 in England

Site-specific biotinylation of purified proteins using BirA

(BioLegend, USA), biotin anti-IgG1 (A85-1), biotin anti-IgG2a[b] (IgG2c) (5.7). All data were 664 collected on an LSR II cytometer (Becton Dickinson, USA) and analyzed with FlowJo software 665 (TreeStar, USA) . 666

Mouse splenocytes were incubated in anti-IFNγ (clone AN-18, Invitrogen) pre-coated plates at 668 37˚C for 20 hours with stimulating peptides or protein. Parallel wells were incubated with 669 vehicles as the negative control or PMA/ionomycin as the positive control (data not shown). 670Pools of 15-mer peptides derived from the RBD sequence or the intact RBD protein were added 671 at 10 μg/ml for stimulation. Pool 1 contains the peptides corresponding to the amino acids 420-672 434, 426-440 and 445-459 in the full-length SARS-CoV-2 S protein, and Pool 2 contains the 673 peptides corresponding to the amino acids 511-525, 517-531 and 535-549 of S protein 31 . Biotin-674 labeled IFNγ antibody (clone R4-6A2, Invitrogen) and subsequent HRP-conjugated streptavidin 675 (Jackson ImmunoResearch, USA) were used for detection. Spots were scanned and counted by 676ImmunoSpot analyzer (Cellular Technology Limited, USA). 677

Splenocytes and bone marrow cells from mice were incubated in RBD protein pre-coated plates 679 at 37˚C for 5 hours. HRP-conjugated anti-mouse IgG (Bethyl Laboratories), anti-mouse IgG1, 680 anti-mouse IgG2b, or anti-mouse IgG2c (Southern Biotech) were used for detection. Spots were 681 scanned and counted by ImmunoSpot analyzer (Cellular Technology Limited, USA). 682

Mouse spleen sections at 7μm thickness were stained with biotinylated anti-IgD (clone 11-26C, 684Invitrogen) and FITC anti-GL-7 (clone GL7, BioLegend) and then with streptavidin-conjugated 685 peroxidase and alkaline phosphatase-conjugated anti-FITC as previously described 33 . The 686 sections were then developed with 3,3'-diaminobenzidine and Fast Red (both from Sigma-687 Aldrich). 688

Four rhesus macaques were injected intramuscularly with 20μg of AP205-RBD twice 3 weeks 690 apart. The other four rhesus macaques were injected with PBS in parallel. Sera were collected at 691 day 14 and 21 after the first immunization and day 7 after the second immunization. 12 days 692 J o u r n a l P r e -p r o o f 26 after the second immunization, SARS-CoV-2 virus (strain 107, provided by the Guangdong 693Provincial Center for Disease Control and Prevention, Guangdong, China) 37 cultured in Vero-E6 694 cells at 5x10 6 median tissue culture infective dose (TCID50) per milliliter were given to the 695 animals via a combination of intranasal (0.4ml / nostril) and intratracheal (1.2ml, fiberoptic 696 bronchoscopy) instillation, so that a total of 1x10 7 TCID50 virus particles were used in the viral 697 challenge for each animal. Body temperature, weight and blood cell counts were monitored at 698 day0, 1, 3, 5, 7 after viral challenge and no significant changes were found for both immunized 699 and control groups of animals. Nasal swab, throat swab and rectal swab samples were taken at 700 day0, 1, 3, 5, 7 after viral challenge and examined for viral load. Tracheal brush samples were 701 taken at day 3 after viral challenge and examined for viral load. Euthanasia was performed on the 702 day 7 after the challenge and tissues from each of the seven lung lobes were taken for viral load 703 and histology examination.