key: cord-0699924-q06qne6e authors: Liang, Joshua G.; Su, Danmei; Song, Tian-Zhang; Zeng, Yilan; Huang, Weijin; Wu, Jinhua; Xu, Rong; Luo, Peiwen; Yang, Xiaofang; Zhang, Xiaodong; Luo, Shuangru; Liang, Ying; Li, Xinglin; Huang, Jiaju; Wang, Qiang; Huang, Xueqin; Xu, Qingsong; Luo, Mei; Huang, Anliang; Luo, Dongxia; Zhao, Chenyan; Yang, Fan; Han, Jian-Bao; Zheng, Yong-Tang; Liang, Peng title: S-Trimer, a COVID-19 subunit vaccine candidate, induces protective immunity in nonhuman primates date: 2020-09-24 journal: bioRxiv DOI: 10.1101/2020.09.24.311027 sha: 92941ce0b57e8510198bd7a0a5f9f0d97d658c63 doc_id: 699924 cord_uid: q06qne6e SARS-CoV-2 is the underlying cause for the COVID-19 pandemic. Like most enveloped RNA viruses, SARS-CoV-2 uses a homotrimeric surface antigen to gain entry into host cells. Here we describe S-Trimer, a native-like trimeric subunit vaccine candidate for COVID-19 based on Trimer-Tag technology. Immunization of S-Trimer with either AS03 (oil-in-water emulsion) or CpG 1018 (TLR9 agonist) plus alum adjuvants induced high-levels of neutralizing antibodies and Th1-biased cellular immune responses in animal models. Moreover, rhesus macaques immunized with adjuvanted S-Trimer were protected from SARS-CoV-2 challenge compared to vehicle controls, based on clinical observations and reduction of viral loads in lungs. Trimer-Tag may be an important new platform technology for scalable production and rapid development of safe and effective subunit vaccines against current and future emerging RNA viruses. 19 convalescent sera and naï ve sera, implying prior infection by influenza in all subjects tested but only SARS-CoV-2 infection in the COVID-19 convalescent subjects. These results support the specificity of the assay and demonstrate the ability of S-Trimer to detect SARS-CoV-2 Spike protein-specific antibodies in convalescent sera, further confirming the native-like conformation of the Spike antigen in S-Trimer. Analysis of antibody titers detected in the convalescent sera was stratified based on various factors (including COVID-19 disease severity, patient age and patient gender) in 34 subjects for whom such information was available ( Fig. 2B and table S1 ). Antibody titers were observed to correlate with disease severity, with lower antibody titers observed in patients with mild COVID-19 disease and higher titers in severe cases, consistent with findings from other published studies (23, 24) . ACE2-competitive antibody titers were detectable in only 33% (n = 3/9) of patients with mild disease, while these antibodies were present in 82% (n = 14/17) of patients with moderate disease and in 100% (n = 7/7) in patients with severe disease. Antibody titers also appeared to moderately correlate with patient age, but no differences were observed between genders. Antibody titers in human convalescent sera were observed to be correlated between the three assays utilized (Fig. 2C ), and these correlations were further confirmed in sera from animals immunized with S-Trimer ( fig. S5 and table S2 ). Interestingly, several convalescent sera samples with detectable pseudoviral neutralizing antibody titers did not have any detectable ACE2-competitive titers (Fig. 2C, right), suggesting that RBD, which binds to ACE2, is not the only target for neutralizing antibodies, and other domains such as NTD and S2 may also be important antigenic epitopes for viral neutralization as previously reported (25, 26) . The immunogenicity of S-Trimer was first evaluated in BALB/c mice. Mice were vaccinated intramuscularly twice in a two-dose prime-boost regimen (Days 0 and 21) with S-Trimer either nonadjuvanted or with various adjuvants including AS03, CpG 1018, and CpG 1018 plus alum. The adjuvant effects on humoral immunogenicity were evident, as S-Trimer binding antibody titers, ACE-2 competitive titers and neutralizing antibody titers in the adjuvanted groups were significantly higher than nonadjuvanted vaccine at corresponding antigen dose levels (Fig. 3 ,A to C). High levels of neutralizing antibody titers were only observed in AS03 and CpG 1018 plus alum adjuvanted groups (Fig. 3C ), but not for non-adjuvanted S-Trimer nor with CpG 1018 alone-adjuvanted S-Trimer. S-Trimer adjuvanted with either AS03 or CpG 1018 plus alum elicited both ACE2-competitve and pseudovirus neutralizing antibody titers similar to or higher than levels observed in human convalescent sera samples. Similar results were observed in rats immunized with S-Trimer ( fig. S6 ), albeit at higher overall antibody titers 7 than in the mouse studies likely in large-part due to the administration of adjuvants (AS03, CpG 1018, CpG 1018 plus alum) at intended-human dose-levels (10-fold higher for AS03 and 75-to 150-fold higher for CpG 1018 compared to doses used in mouse studies). S-Trimer antigen-specific cell-mediated immunity (CMI) was studied by harvesting splenocytes from immunized mice at sacrifice, followed by stimulation with S-Trimer antigen and detection of Th1 (IL-2 and IFN) and Th2 (IL-4 and IL-5) cytokines by ELISpot. The CpG 1018 plus alum and AS03 groups appeared to induce a stronger overall CMI response than non-adjuvanted S-Trimer (Fig. 3D) . A Th1-biased cell-mediated immune response was observed across non-adjuvanted and CpG 1018adjuvanted (with or without alum) S-Trimer groups, while a mixed Th1-Th2 profile was observed for AS03. CMI did not appear to be dependent on the dose of antigen ( fig. S7 ). The immunogenicity of adjuvanted S-Trimer was further studied in nonhuman primates (rhesus macaques). Animals (n = 6 per group) were vaccinated intramuscularly twice (at Day 0 and 21) with AS03-adjuvanted S-Trimer, CpG 1018 plus alum-adjuvanted S-Trimer, or a PBS vehicle control. The animals were then challenged on Day 35 with 2.6 x 10 6 TCID50 (60% intratracheal and 40% intranasal) SARS-CoV-2 virus and then evaluated for immune protection by various parameters. High levels of binding and neutralizing antibody titers measured by different methods, including wild-type SARS-CoV-2 virus neutralization assay, were observed in both groups receiving adjuvanted S-Trimer (Fig. 4 ,A to D). The boost-effect of the second dose (on Day 21) was evident, with significant increases in neutralizing antibody levels observed at Day 28 and continuing to rise through Day 35 prior to challenge. At Day 35, neutralizing antibody titers in the AS03-adjuvanted S-Trimer group were significantly higher than levels in human convalescent sera (Fig. 4) . For animals in the CpG 1018 plus alum group, despite exhibiting numerically lower binding and neutralizing antibody titers than the AS03adjuvanted S-Trimer group, levels of antibodies were still within the range of human convalescent sera ( Fig. 4) . Moreover, animals in the CpG 1018 plus alum group also appeared to mount a rapid and more durable lymphocyte response that remained high 7 days after viral challenge, compared to AS03 and vehicle groups ( fig. S8 ). Interestingly, antibody titers post-viral challenge appeared to modestly decrease following challenge at Day 40 (5 days post inoculation [dpi]), suggesting that challenge with high doses of SARS-CoV-2 may have led to rapid binding of circulating anti-Spike antibodies to the virus and subsequent clearance; a similar trend was reported in convalescent humans that were re-exposed to the virus (27) . Following challenge with SARS-CoV-2, animals in the adjuvanted S-Trimer groups were protected from body weight loss, whereas animals in the vehicle control group observed rapid body weight loss of approximately 8% through 7 dpi ( Fig. 5A and fig. S9 ), in line with other reported studies (28) . Similarly, animals receiving adjuvanted S-Trimer appeared to be protected from increases in body temperature following SARS-CoV-2 challenge (Fig. 5B) . Various blood chemistry parameters also suggested that animals in the active vaccine groups may have been protected from organ and tissue damage and other adverse effects of SARS-CoV-2 infection ( fig. S10 ), as animals in the vehicle control group observed increases in blood albumin (ALB), A/G ratio, AST, creatine kinase (CK), glucose (GLU), lactic acid (LAC), and triglycerides (TRIG) through 7 dpi compared to the adjuvanted S-Trimer groups. Lung tissues were harvested at necropsy from 5 to 7 dpi and tested for SARS-CoV-2 viral loads based on genomic RNA (gRNA). Complete reduction of viral loads in lung tissues was observed in AS03 and CpG 1018 plus alum adjuvanted S-Trimer groups, whereas viral loads were detectable in the vehicle group ( Fig. 5C and fig. S11 ). Similar trends of reduced viral loads in animals receiving active vaccine were observed from throat swabs, anal swabs and tracheal brushes after challenge through 7 dpi (Fig. 5D ). Viral gRNA detected in nasal swabs were expected given the location of viral challenge and is not necessarily indicative of replicating virus. Histopathological analysis conducted in lung tissues and IHC staining with antibody specific to the Spike protein further confirmed the reduced SARS-CoV-2 infection in animals vaccinated with S-Trimer (Fig. 5E ). Since SARS-CoV-2 with D614G mutation in the Spike protein has become the predominant circulating strain in many regions of the world (29), we also produced S-Trimer with the D614G mutation. The results showed that, compared to the wild-type S-Trimer, no significant differences were observed in ACE2 binding affinity, nor ACE2 competitive binding against anti-Spike neutralizing antibodies produced from animals immunized with wild-type S-Trimer ( fig. S12 ). Unlike other full-length Spike proteins previously used for structural studies and vaccine development that utilized mutations introduced to abolish S1/S2 cleavage by furin protease and reportedly stabilize the protein in a prefusion form (6, 30) , S-Trimer is partially cleaved at the S1/S2 junction, similar to S 9 proteins isolated from live SARS-CoV-2 virus (31) and recombinant full-length S expressed in HEK293 cells (32) . Importantly, we demonstrated that the S-Trimer vaccine candidate, with a fully wild-type S sequence from SARS-CoV-2, is not only expressed at high levels in CHO cells but also is highly glycosylated and adopts a native-like trimeric pre-fusion conformation. N-terminal protein sequence analysis revealed that upon signal peptide removal during its biosynthesis, S-Trimer has N-terminal 14Q modified by pyroglutamate formation to protect itself from exo-protease degradation, suggesting that S protein from SARS-CoV-2 can be very stable in vivo. Fusion to Trimer-Tag allows the soluble wild-type S protein to form a disulfide bond-linked homotrimer with a partially-cleaved S1 that remains noncovalently bound to S-Trimer and also to maintain high affinity binding to the ACE2 receptor, thus preserving the crucial antigenic epitopes necessary for viral neutralization. In addition to tailored-affinity purification scheme we developed for any Trimer-Tagged fusion proteins followed by further downstream purification steps typical for the production of modern biologics to ensure purity and safety (including preventative VI and VR), the current titer in bioreactor of approximately 500 mg/L would predict that billions of doses of S-Trimer antigen to be used in a COVID-19 vaccine may be produced annually from several 2,000L bioreactors. The potential production output of S-Trimer antigen further supports the rationale to advance its clinical development with multiple adjuvants in parallel (such as AS03 and CpG 1018 plus alum), should supply of a single adjuvant be a limiting factor for the global supply of the vaccine (antigen plus adjuvant). A Phase I clinical trial is currently ongoing to evaluate the safety and immunogenicity of S-Trimer with AS03 and CpG 1018 plus Alum adjuvants (NCT04405908). Understanding which components of the immune system are needed to confer optimal immune protection against SARS-CoV-2 infection and COVID-19 disease is critical to the development of effective vaccines. It has been reported that individuals with mild COVID-19 disease observe low or undetectable levels of neutralizing antibodies (23, 24) , and approximately 35% of SARS-CoV-2 naï ve individuals have cross-reactive CD4 + T-cell responses to SARS-CoV-2 antigens due to prior infection by other commoncold coronaviruses (33) . In this study, a clear association between higher antibody titers specific to SARS-CoV-2 and more severe disease was observed, using a panel of 41 human convalescent sera samples collected from recovered COVID-19 patients. In fact, most patients with mild COVID-19 disease did not have any detectable ACE2-competitive titers and only had low neutralizing antibody titers (Fig. 2B ), implying that a strong neutralizing humoral immune response may not be the only component of the immune system that is involved in the prevention or recovery from COVID-19 disease. These observations suggest that SARS-CoV-2 could be particularly susceptible to cell-mediated immune responses, and some patients that develop rapid adaptive T-cell responses may not additionally need high levels of neutralizing antibodies to eliminate the virus. Indeed, it has been reported that patients with asymptomatic or mild COVID-19 disease develop robust T-cell immunity, while patients with severe COVID-19 disease observed T-cells at unphysiologically low levels (34) and potentially require higher levels of neutralizing antibodies in order to mount an effective recovery. While neutralizing monoclonal antibodies against SARS-CoV-2 Spike protein have been demonstrated to be protective against viral challenge in animals (35, 36) , it appears likely that COVID-19 vaccines inducing both humoral and cellmediated immune responses may confer optimal protection against SARS-CoV-2. Vaccine adjuvants can contribute to achieving stronger immune responses to a viral antigen. The use of an adjuvant is of particular importance in a pandemic situation, since it could reduce the amount of antigen required per dose, allowing significantly more doses of vaccine to be produced and therefore contributing to the protection of more people. Importantly, AS03, CpG 1018 and alum adjuvants have all been utilized in commercially-licensed vaccines and have significant safety databases in clinical and postmarketing studies (37, 38) . In our studies, we have observed significant adjuvant effects of AS03 and CpG 1018 plus alum, with robust high-level induction of both humoral and cell-mediated immune responses to S-Trimer in rodents and nonhuman primates. Interestingly, we did observe some differences in the immune responses stimulated by these two adjuvant systems. In nonhuman primates, AS03 appeared to induce a stronger humoral immune response, inducing higher levels of neutralizing antibody titers than CpG 1018 plus alum. While antibody titers were lower in nonhuman primates for CpG 1018 plus alum (albeit still in the range of or higher than human convalescent sera), CpG 1018 plus alum did appear to potentially induce a durable cellular immune response (as measured by lymphocyte frequency) in nonhuman primates and was more strongly Th1-biased in rodents. However, there were no clear differences in the immune protection against SARS-CoV-2 challenge observed between the two adjuvant systems in nonhuman primates, suggesting that both adjuvants had induced sufficient and protective levels of immunity. Importantly, no signs of disease enhancement were observed, a theoretical concern for SARS-CoV-2 vaccines based on prior experience with vaccine candidates against SARS-CoV and RSV that utilized inactivated viruses (11, 12) . Our data demonstrate that S-Trimer adjuvanted with either AS03 or CpG 1018 plus alum can induce robust humoral and cellular immune responses in various animal species and protective immunity against SARS-CoV-2 infection in nonhuman primates, with no signs of disease enhancement. 11 Collectively, these results support the advancement of adjuvanted S-Trimer through human clinical studies to further demonstrate safety, immunogenicity, and vaccine efficacy. Importantly, the recombinant production of S-Trimer utilizing Trimer-Tag technology has been streamlined with the ability to be rapidly scaled-up to billions of doses annually, and the subunit vaccine can be stored at 2-8°C (does not require frozen storage conditions). These advantages could allow S-Trimer vaccine candidate, if successful in clinical studies, to contribute significantly to the control of the COVID-19 pandemic. A Phase 1 clinical study was initiated in June 2020 (NCT04405908), and late-stage clinical studies to evaluate vaccine efficacy and safety are also planned. Should adjuvanted S-Trimer be proven successful as a COVID-19 subunit vaccine, Trimer-Tag may become an important new platform technology for rapid responses to future threats posed by any emerging enveloped RNA viruses. The authors would like to acknowledge and thank GSK and Dynavax Technologies Corporation for providing AS03 and CpG 1018 adjuvants, respectively, for this study. We thank Lihong Chen and Xiaojun Huang at Institute of Biophysics, Chinese Academy of Sciences for assistance in conducting the negative EM studies. We would also like to thank Xiaodong Wang for helpful discussions for this study and critical review and comments for this manuscript. Figs. S1 to S12 Tables S1 to S2 To produce the wild-type secreted S-Trimer fusion protein, a cDNA encoding the ectodomain of wild-type SARS-CoV-2 spike (S) protein (amino acid residues 1 to 1211) (GenBank: After harvesting the clarified cell culture medium via depth-filtration (Millipore) to remove cells, S-Trimer was purified to homogeneity by consecutive chromatographic steps. A Protein A affinity column using MabSelect PrismA (GE Healthcare) preloaded with Endo180-Fc was used to affinity-capture S-Trimer, based on the high affinity binding between Endo180 and Trimer-Tag (19) (fig. S1 ). After washing off unbound contaminating proteins, S-Trimer was eluted using 0.5 M NaCl in phosphate buffered saline, conditions that do not elute Endo180-Fc from Protein A. After one hour of low pH (pH 3.5) viral inactivation (VI) using acetic acid, the pH was adjusted to neutral range before loading onto Capto QXP resins (GE BioSceinces) in a flow-through mode to remove host cell DNA and residual host cell proteins (HCP). Then, a preventative viral removal (VR) step using nanofiltration followed by a final UF/DF (Millipore) for buffer change were used to achieve the S-Trimer active drug substance (DS). The purity of S-Trimer was analyzed by Size-Exclusion Chromatography (SEC-HPLC) using Agilent 1260 Infinity HPLC with an analytic TSK gel G3000 SWxL column (Tosoh). Phosphate Buffered Saline (PBS) was used as the mobile phase with OD280 nm detection over a 20 min period at a flow rate of 1 ml/min. The binding affinity of S-Trimer to ACE2 was assessed by Bio-Layer Interferometry measurements on ForteBio Octet QKe (Pall). ACE2-Fc (10 µg/mL) was immobilized on Protein A (ProA) biosensors (Pall). Real-time receptor-binding curves were obtained by applying the sensor in two-fold serial dilutions of S-Trimer (22.5-36 µg/mL in PBS). Kinetic parameters (Kon and Koff) and affinities (KD) were analyzed using Octet software, version 12.0. Dissociation constants (KD) were determined using steady state analysis, assuming a 1:1 binding model for a S-Trimer to ACE2-Fc. S-Trimer was diluted to 25 ug/mL in PBS with pH adjusted to 5.5 with acetic acid and applied for 1 min onto the carbon-coated 400 CU mesh grid that had been glow-discharged at 12mA for 20s. The grids were negatively stained with 1% (w/v) uranyl formate at pH 4.0 for 1 min. The samples were collected through FEI Tecnai spirit electron microscope operating at 120 KeV. S-Trimer binding antibody titers (or HA-Trimer binding antibody titers) in sera samples collected from immunized animals was determined by ELISA. 96-well plates (Corning) were coated with S-Trimer (or HA-Trimer) (1μg/mL, 100 μL/well) at 4℃ overnight and blocked with 2% non-fat milk at 37℃ for 2 h. Serial dilutions of the antisera were added to the wells. After incubating for 1h at 37℃, the plates were washed 3 times with PBST (PBS containing 0.05% Tween-20), followed by incubating with goat anti-mouse, rat, monkey, or human IgG-HRP (Southern Biotech) at 37℃ for 30 min. Plates were then washed 3 times with PBST and signals were developed using TMB substrate (Thermo Scientific). The colorimetric reaction was stopped after 5 min by adding 2M HCl. The optical density (OD) was measured at 450 nm. Antibody titers (EC50) were defined as the reciprocal of the dilution from a sample at which 50% of the maximum absorbance was observed. A modified logit-log equation ("Fit Equation" ) was used to fit serum titration data for EC50 determination. 96-well plates (Corning) were coated with 1 μg/mL ACE2-Fc (100 μL/well) at 4℃ overnight, blocked with 2% non-fat milk 37℃ for 2 h. After washing 3 times with PBST, the plates were incubated with S-Trimer (100 ng/mL) mixed with serially diluted antisera for 1 h at 37℃. After SARS-CoV-2 pseudovirus neutralization assay was conducted as previously described (40) The animal model for SARS-CoV-2 challenge in rhesus macaques was conducted as previously described (41, 42) dpi, including body weight and body temperature. Tracheal brushing and various swabs (throat and anal) were collected 3, 5, and 7 dpi, and total RNA was extracted for viral load analysis by qRT-PCR. At 5 dpi, 2 rhesus macaques per group were euthanized, and at 7dpi, the remaining animals were euthanized. Lung tissue samples were homogenized, and total RNA was extracted for viral load analysis by qRT-PCR. For detection of SARS-CoV-2 genomic RNA, primers and probes used in this experiment included: forward primer 5'-GGGGAACTTCTCCTGCTAGAAT-3', reverse primer 5'-CAGACATTTTGCTCTCAAGCTG-3', and probe FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3', as previously described (41, 42) . Pathological examination by H&E staining and IHC staining with a mouse monoclonal antibody specific for S1 of SARS-CoV-2 (Clover Biopharma) were conducted. Wild-type SARS-CoV-2 neutralization assay was performed in the BSL-3 lab at Kunming Institute of Zoology, CAS. Vero-E6 cells (2×10 4 per well) were seeded in a 96-well plate overnight. On a separate 96-well plate plate, heat-inactivated antisera (56°C for 30 min) from Rhesus Macaques were serially diluted in cell culture medium in 3-fold dilutions starting from 1:100. The diluted sera were mixed with an equal volume of solution containing 100 TCID50 live SARS-CoV-2 virus in each well. After 1 h incubation at 37°C in a 5% CO2 incubator, the virusserum mixtures were transferred to the 96-well plate containing Vero-E6 cells and cultured in a 5% CO2 incubator at 37°C for 6 days. Cytopathic effect (CPE) of each well was recorded under microscopes, and the neutralization titer (MN50) was calculated as the reciprocal of serum dilution required for 50% neutralization of viral infection. Lung tissues collected from the Rhesus Macaques after SARS-COV-2 viral challenge were fixed in 10% formalin and paraffin embedded. Sections (5 µm) were prepared and stained with hematoxylin and eosin (H&E). For SARS-CoV-2 viral detection, immunohistochemical staining for SARS-CoV2-S antigen was carried out by incubating a mouse monoclonal antibody specific to S1 of SARS-CoV-2 (Clover Biopharma) overnight at 4°C . After washing with PBST, HRPconjugated secondary antibody (ZSGB Bio PV-6002) was added for 1h at room temperature, the sections were developed with DAB (ZSGB Bio ZLI-9017), and mounted with Neutral Balsam for analysis under an upright microscope (BX53, Olympus). Statistical analyses were performed using the Prism 8.0 (GraphPad Software). Two-sided Mann-Whitney tests were used to compare two experiment groups. Comparisons among multiple groups were performed using Kruskal-Wallis ANOVA with Dunn's multiple comparisons tests. Twoway ANOVA test with Tukey's multiple comparison test was applied for multiple groups at different time points. P values < 0.05 were considered significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS, no significance. yield peptide sequence shown with predicted furin protease cleavage at S1/S2 boundary. Following the removal of 14Q by pyroglutamate aminopeptidase, the N-terminal sequence for the full-length S-Trimer was determined by subsequent Edman degradation as indicated. Since the S2-Trimer had predicted Nterminus after furin cleavage, the N-terminus of S1 was predicted to be the same as that of full-length S-Trimer. virus. Following SARS-CoV-2 challenge, various hematology parameters were analyzed at 0, 1, 3, 5 and 7 dpi. All data are presented as mean ± SEM. Table S2 . Correlation of antibody titers in immunized mice, rats, rhesus and human convalescent sera. Antibody titers in human convalescent sera and in immunized mice, rats, and rhesus macaques based on three assays (S-Trimer binding antibodies, ACE2-competitive, and pseudovirus neutralization) were analyzed for correlation based on Pearson's R. Estimates of global seasonal influenzaassociated respiratory mortality: a modelling study influenza: the mother of all pandemics Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China Middle East Respiratory Syndrome Coronavirus (MERS-CoV) origin and animal reservoir Coronavirus Resource Center Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation A pneumonia outbreak associated with a new coronavirus of probable bat origin Understanding novel COVID-19: its impact on organ failure and risk assessment for diabetic and cancer patients Developing Covid-19 vaccines at pandemic speed A strategic approach to COVID-19 vaccine R&D Immunopathogenesis associated with formaldehyde-inactivated RSV vaccine in preclinical and clinical studies A double-inactivated Severe Acute Respiratory Syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge HIV-1 envelope glycoprotein immunogens to induce broadly neutralizing antibodies The modern era of HIV-1 vaccine development Human immunodeficiency virus type 1 neutralization is determined by epitope exposure on the gp120 oligomer HIV vaccine design and the neutralizing antibody problem HIV-1 envelope trimer design and immunization strategies to induce broadly neutralizing antibodies Improvement of pharmacokinetic profile of TRAIL via Trimer-Tag enhances its antitumor activity in vivo Endo180 binds to the C-terminal region of type I collagen A urokinase receptor-associated protein with specific collagen binding properties Endoprotease activities other than furin and PACE4 with a role in processing of HIV-I gp160 glycoproteins in CHO-K1 cells Disease severity dictates SARS-CoV-2-specific neutralizing antibody responses in COVID-19 Neutralizing antibody responses to Severe Acute Respiratory Syndrome Coronavirus 2 in Coronavirus Disease 2019 inpatients and convalescent patients A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2 Perspectives on therapeutic neutralizing antibodies against the novel Coronavirus SARS-CoV-2 Neutralizing antibodies correlate with protection from SARS-CoV-2 in humans during a fishery vessel outbreak with high attack rate Respiratory disease in rhesus macaques inoculated with SARS-CoV-2 Making sense of mutation: what D614G means for the COVID-19 pandemic remains unclear SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 elicits immunogenicity in baboons and protection in mice Development of an inactivated vaccine candidate for SARS-CoV-2 Distinct conformational states of SARS-CoV-2 spike protein SARS-CoV-2-reactive T cells in healthy donors and patients with COVID-19 Karolinska COVID-19 Study Group Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19 Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model SARS-CoV-2 neutralizing human antibodies protects against lower respiratory tract disease in a hamster model Development and evaluation of AS03, an Adjuvant System containing αtocopherol and squalene in an oil-in-water emulsion Development of the CpG adjuvant 1018: a case study Adjuvant System AS03 containing α-tocopherol modulates innate immune response and leads to improved adaptive immunity Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2 Respiratory disease in rhesus macaques inoculated with SARS-CoV-2 Delayed severe cytokine storm and immune cell infiltration in SARS-CoV-2-infected aged Chinese rhesus macaques References and Notes Estimates of global seasonal influenza-associated respiratory mortality: a modelling study influenza: the mother of all pandemics Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China Middle East Respiratory Syndrome Coronavirus (MERS-CoV) origin and animal reservoir Coronavirus Resource Center Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation A pneumonia outbreak associated with a new coronavirus of probable bat origin Understanding novel COVID-19: its impact on organ failure and risk assessment for diabetic and cancer patients Developing Covid-19 vaccines at pandemic speed A strategic approach to COVID-19 vaccine R&D Immunopathogenesis associated with formaldehyde-inactivated RSV vaccine in preclinical and clinical studies A double-inactivated Severe Acute Respiratory Syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge HIV-1 envelope glycoprotein immunogens to induce broadly neutralizing antibodies The modern era of HIV-1 vaccine development Human immunodeficiency virus type 1 neutralization is determined by epitope exposure on the gp120 oligomer HIV vaccine design and the neutralizing antibody problem HIV-1 envelope trimer design and immunization strategies to induce broadly neutralizing antibodies Improvement of pharmacokinetic profile of TRAIL via Trimer-Tag enhances its antitumor activity in vivo Endo180 binds to the C-terminal region of type I collagen A urokinase receptor-associated protein with specific collagen binding properties Endoprotease activities other than furin and PACE4 with a role in processing of HIV-I gp160 glycoproteins in CHO-K1 cells Disease severity dictates SARS-CoV-2-specific neutralizing antibody responses in COVID-19 Neutralizing antibody responses to Severe Acute Respiratory Syndrome Coronavirus 2 in Coronavirus Disease 2019 inpatients and convalescent patients A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2 Perspectives on therapeutic neutralizing antibodies against the novel Coronavirus SARS-CoV-2 Neutralizing antibodies correlate with protection from SARS-CoV-2 in humans during a fishery vessel outbreak with high attack rate Respiratory disease in rhesus macaques inoculated with SARS-CoV-2 Making sense of mutation: what D614G means for the COVID-19 pandemic remains unclear SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 elicits immunogenicity in baboons and protection in mice Development of an inactivated vaccine candidate for SARS-CoV-2 Distinct conformational states of SARS-CoV-2 spike protein SARS-CoV-2-reactive T cells in healthy donors and patients with COVID-19 Karolinska COVID-19 Study Group Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19 Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model SARS-CoV-2 neutralizing human antibodies protects against lower respiratory tract disease in a hamster model Development and evaluation of AS03, an Adjuvant System containing α-tocopherol and squalene in an oil-in-water emulsion Development of the CpG adjuvant 1018: a case study Adjuvant System AS03 containing αtocopherol modulates innate immune response and leads to improved adaptive immunity Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2 Respiratory disease in rhesus macaques inoculated with SARS-CoV-2 Delayed severe cytokine storm and immune cell infiltration in SARS-CoV-2-infected aged Chinese rhesus macaques Table S1 . Human convalescent sera panel. Characteristics and information of 41 COVID-19 patients, from whom convalescent sera was collected and included for testing in this study. COVID-19 disease severity, patient age and patient gender are included, as well as S-Trimer binding antibody titers (EC50), ACE2-competitive titers (EC50) and SARS-CoV-2 pseudovirus neutralization titers (EC50).