key: cord-1004057-5i1odl26 authors: Richmond, P.; Hetchual, L.; Dong, M.; Ma, B.; Hu, B.; Smolenov, I.; Li, P.; Liang, P.; Han, H. H.; Liang, J.; Clemens, R. title: A first-in-human evaluation of the safety and immunogenicity of SCB-2019, an adjuvanted, recombinant SARS-CoV-2 trimeric S-protein subunit vaccine for COVID-19 in healthy adults; a phase 1, randomised, double-blind, placebo-controlled trial date: 2020-12-04 journal: nan DOI: 10.1101/2020.12.03.20243709 sha: 1684345409d25f44906b8f9776db08bf1a4b7175 doc_id: 1004057 cord_uid: 5i1odl26 Background: As part of the accelerated development of prophylactic vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) we report a first-in-human dose-finding and adjuvant justification study of SCB-2019, a novel protein subunit vaccine candidate composed of a stabilised trimeric form of the spike (S)-protein produced in CHO-cells, combined with two different adjuvants. Methods: This phase 1 study was done in one centre in Western Australia in 151 healthy adult volunteers in two age groups (18 to 54 and 55 to 75 years), allocated to 15 groups (nine young and six older adults) to receive two doses, 21 days apart, of placebo, or 3 {micro}g, 9 {micro}g or 30 {micro}g SCB-2019, alone or adjuvanted with AS03 or CpG/Alum. Reactogenicity was assessed for 7 days after each vaccination. Humoral responses were measured as SCB-2019 binding and ACE2-competitive binding IgG antibodies by ELISA, and as neutralising antibodies by wild-type SARS-CoV-2 microneutralisation assay; cellular responses to pooled S-protein peptides were measured by flow-cytometric intracellular cytokine staining. Findings: We report on 148 participants with at least 4 weeks follow-up post dose 2. Three participants withdrew, two for personal reasons and one with an unrelated SAE (pituitary adenoma). Vaccination was well tolerated, with few Grade 3 solicited adverse events (AE). Most local AEs were mild injection site pain, which were more frequent with formulations containing AS03 than CpG/Alum or unadjuvanted SCB-2019. Systemic AEs, mostly transient headache, fatigue or myalgia, were more frequent in young adults than older adults after the first dose, but similar after second doses. Unadjuvanted SCB-2019 elicited minimal immune responses, but SCB-2019 with fixed doses of AS03 or CpG/Alum induced high titres and seroconversion rates of binding and neutralising antibodies in both young and older adults. Titres were higher than those observed in a panel of COVID-19 convalescent sera in all AS03 groups and high dose CpG/Alum groups. Both adjuvanted formulations elicited Th1-biased CD4+ T cell responses. Interpretation: SCB-2019 trimeric protein formulated with AS03 or CpG/Alum adjuvants elicited robust humoral and cellular immune responses against SARS-CoV-2 with high viral neutralising activity. Both adjuvanted formulations were well tolerated and are suitable for further clinical development. To date, the global COVID-19 pandemic due to the SARS-CoV-2 virus has caused 65 million infections and almost 1·5 million deaths [1] . Infections are leading to unprecedented numbers of cases of severe respiratory illness with significant proportions of patients requiring admission to intensive care units (ICU) [2] . COVID-19 is associated with a high transmission rate and without adequately effective therapies rising numbers of cases of respiratory distress are threatening to overwhelm global healthcare capacity. Interventions are urgently required to reduce this disease burden leading to the accelerated introduction into clinical development of at least 47 vaccine candidates [3] . The main viral antigenic target is the glycosylated Spike (S) protein, a trimeric protein consisting of two subunits, S1 and S2 [4] , which is an essential component for viral binding, fusion and uptake into mammalian cells [5] . S1 interacts with the receptor binding domain (RBD) of human cell-surface human angiotensin-converting enzyme 2 (ACE2) and following proteolytic cleavage of the two subunits the S2 domain undergoes a major conformational change which leads to fusion and intracellular uptake of the viral mRNA for replication [6, 7] . SARS-CoV-2 in vivo in animal challenge studies [11] . This first-in-human phase 1 dosefinding and adjuvant justification study was performed to assess the safety, tolerability and immunogenicity of three dose levels of SCB-2019 when administered to healthy adults as two doses 21 days apart without adjuvant or formulated with either AS03 or CpG/Alum. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.20243709 doi: medRxiv preprint We report the interim analysis of the first stage of a phase 1 randomized, double-blind, placebo-controlled study of SCB-2019. The trial was done in one study centre in Nedlands, Western Australia, Australia from June 19, 2020 until database lock on October 20, 2020. The study protocol was approved by the study centre Institutional Review Board and registered with ClinicalTrials.gov (identifier NCT04405908). The study was done according to International Conference of Harmonisation and Good Clinical Practice guidelines. The overall objectives were to assess the safety, tolerability and immunogenicity of three increasing dosages of SCB-2019, unadjuvanted or adjuvanted with AS03 or CpG/Alum, in young and older adults when administered as two intramuscular doses 21 days apart. This first stage was a placebo-controlled dosage escalation study done in two parts with 15 groups of 10 participants each. The first part was done in nine groups of young adults years inclusive) using a sentinel strategy in which the first two participants assigned to each group (one vaccinee, one placebo control) received their study injections. Sentinels were monitored for 48 hours and the safety data reviewed by a Safety Monitoring Committee (SMC) to assess any significant AEs that occurred before the remaining eight participants of that group (seven vaccinees, one placebo) were treated. No sentinel strategy was applied to the second part of the study performed in six groups of older adults (55-75 years inclusive) for which recruitment was only started after the safety from the equivalent adult group (same dose and formulation) had been considered by the SMC. Further expansion of the study is planned for long term safety follow-up as well to generate data on antibody persistence, and to include SARS-CoV-2 seropositive participants. Results of those investigations will be reported separately. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.20243709 doi: medRxiv preprint Eligible participants were adults of either sex from 18 to 75 years of age who were healthy at enrolment based on medical history and medical assessment. All volunteers were screened for serum antibodies against SARS-CoV-2 as evidence of prior infection, and for acute exposure using reverse transcriptase-polymer chain reaction (RT-PCR), which were repeated at each study visit. Inclusion criteria included being able to provide informed consent, having a BMI between 18·5 and 35·0 kg/m 2 , being able to understand and sign the informed consent and being available for the duration of the study (6 months) . Female participants of childbearing potential were not to be pregnant or breastfeeding and had to agree to use protocol-approved forms of contraception until 6 months after the first vaccination. Men were also to use a protocol-approved form of contraception from the day of first vaccination until 6 months after the first vaccination and refrain from donating sperm over the same period. Main exclusion criteria included positive serology for SARS-CoV-2, any uncontrolled chronic medical disorders, any known or suspected impairment of the immune system due to known immunosuppressive conditions or any therapy with immunosuppressants or immunostimulants, known allergy to any vaccine components, malignancies, a positive screening serology for HIV, hepatitis B or C, or prior receipt of any other SARS-CoV-2 vaccine. All volunteers were asked to avoid strenuous exercise from screening to Day 50. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.20243709 doi: medRxiv preprint gentle inversion at room temperature for administration within 1 hour. Each 0·5 mL vaccine dose, containing 3 μg, 9 μg or 30 μg SCB-2019 with sodium phosphate buffer and 0·05 mg polysorbate 80 in 0·9% sodium chloride, was withdrawn into a syringe for injection and administered by intramuscular injection in the deltoid region. Placebo was 0.5mL 0·9% sodium chloride for injection. Participants were assigned a study number at enrolment and vaccinated according to a randomisation list prepared by the study sponsor. All participants and personnel involved in safety data collection and immunogenicity assessments were blinded to the study treatment. Vaccine preparation and administration were performed by different unblinded study personnel, using opacified syringes to maintain the participant blind as the vaccine and placebo are visually different. On Day 1, before vaccination, each participant received a full physical examination when vital signs were recorded and a blood sample was drawn for baseline safety laboratory parameters. Further safety blood samples were drawn on Days is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.20243709 doi: medRxiv preprint from Day 1 to Day 50. Serious adverse events (SAE) and adverse events of special interest (AESI) (see Supplementary Appendix page 2) occurring before the database lock were to be reported immediately to the investigator and then to the study sponsor within 24 hours. During study conduct a safety monitoring committee continuously assessed safety data with the option to authorise use of stopping/pausing rules predefined in the protocol. All study participants were tested for SARS-CoV-2 by nasopharyngeal swab for reverse transcription polymerase chain reaction (RT-PCR) at each study visit. If a participant was suspected to be infected with SARS-CoV-2 virus or had confirmed COVID-19 between study visits, the participant was requested to have an additional test for SARS-CoV-2 infection. Blood was drawn to prepare serum samples for immunogenicity assessments before the is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.20243709 doi: medRxiv preprint PBMCs were collected from all participants at Days 1, 22, 36 and 50 to assess T-cell mediated immune responses to vaccination using intracellular cytokine staining (ICS) flow cytometry to measure CD4-positive T cells expressing markers including IFN-γ, IL-2, IL 4, IL-5 and IL-17 after stimulation with SARS-CoV-2 S-protein peptide pools. There was no formal statistical hypothesis in this phase 1 study and all data summaries are presented descriptively by group. The study sample size was not based on any statistical hypothesis but is typical of such phase 1 studies and was considered to be adequate to provide a preliminary assessment of vaccine safety and reactogenicity in each cohort. The Safety Analysis Set (SAS) consists of all subjects randomised to receive at least one dose of study vaccine or placebo, analysed according to the treatment they actually received. Reported summary statistics include counts and percentage of participants who reported at least one solicited local reactions and systemic AEs and unsolicited AEs (with severity and causality), and SAEs and AESIs, after the first and second doses. For this report, the safety data for all participants with at least 21-day safety follow up after Dose 1 are included. The main analysis population for the immunogenicity analysis in this report is the immunogenicity full analysis set, consisting of all participants in the SAS with at least one post vaccination blood sample collected and analysed for immunogenicity. Subjects are summarised according to treatment received. Antibody responses are presented as geometric mean titres (GMT) with 95% confidence intervals (95% CI) at each blood sampling timepoint for each vaccine group. Geometric mean values are calculated on Log10 (titres/data) values, with subsequent antilog transformations applied, the 95% CI being calculated using normal distribution. Seroconversion rates, defined as the percentage of participants with at least a four-fold increase in antibody titre over baseline within each study group, were calculated for . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.20243709 doi: medRxiv preprint the Day 22, 36 and 50 timepoints. For the between group comparisons in geometric means an analysis of variance (ANOVA) model was fitted to log-transformed assessment (such as titre) values based on the subjects with available data at each timepoint then the geometric mean ratio (GMR) and the 95% CI were calculated. Two-sided 95% CIs for the GMR were obtained by calculating CIs using Student's t-distribution for the mean difference of the logarithmically transformed results and antilog transformation of the confidence limits. All analyses, and summaries were on group unblinded data performed using SAS ® software (version 9·4 or higher) or GraphPad Prism, v.6.0c. Authors who are employees or a scientific advisor of the sponsor participated in design and development of the protocol, data analysis and interpretation. The lead author worked with a medical writer financed by the study sponsor to prepare a first draft manuscript which was reviewed and revised by all authors, who also made the decision to submit for publication. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.20243709 doi: medRxiv preprint A total of 329 volunteers were screened, of whom 151 (91 young adults and 60 older adults) were enrolled in their respective age strata after testing negative for SARS-CoV-2 (figure 1). The majority of the screen failures (173 of 178 [97%]) were due to exclusion criteria. One adult assigned to the 30 μg SCB-2019 group who withdrew before receiving any vaccination was replaced with another volunteer. Demographics were similar across groups (table 1). In the young adult groups the mean age of all SCB-2019 recipients was 36·2 ± 11·5 years vs. 32·6 ± 10·7 years for placebo recipients, 40% were male, and most described themselves as white and neither Hispanic nor Latino. In older adults the mean age was 61·1 ± 4·9 years in vaccinees and 62·3 ± 5·9 years in placebo controls, 47% were male, and all were white. There were no deaths or hospitalisations during the study, and only two SAEs both in older adults. One older adult was diagnosed with cellulitis following a cat bite but completed the study, while another had hyponatraemia after receiving one dose in the 9 μg CpG/Alum group and was withdrawn from the study (figure 1). The participant was subsequently found to have a pituitary adenoma, which is a known to potentially cause hyponatremia [12] [13] [14] . Neither event was considered to be associated with vaccination. One adult decided to is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. Young adults reported local AEs more frequently (39% of all dose levels combined) than the older adults (21%) after the first dose, but incidence rates were similar in the two age groups, 35% and 34%, respectively, after the second dose. After the first dose of plain SCB-2019 solicited systemic AEs were infrequent and similar to placebo in the 3 μg and 9 μg groups, but 50% of the 30 μg group reported Grade 1 or 2 AEs. These rates were lower after the second doses (figure 2). In contrast, when formulated with AS03 the frequency of systemic AEs was higher and not dose-dependent, reported by 25-38% per group after the first dose and 44-56% after the second dose with a concomitant increase in the proportion described as Grade 2. Two participants, one each in the 9 μg and 30 μg groups, reported Grade 3 fatigue and myalgia. The most frequently reported systemic adverse events were headache, fatigue, and myalgia, with six reports of fever, all Grade 1 or 2 after the second dose of SCB-2019+AS03. Systemic AE rates in those who received SCB-2019+CpG/Alum were similar to the SCB-2019+AS03 group after the first dose, but there was no consistent trend to increase frequency or severity after the second dose. As with local AEs, the frequency of reported systemic AEs was lower in older adults after their first dose (17%) than the younger adults (38%), and overall rates were similar after second doses, 30% . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.20243709 doi: medRxiv preprint in older and 34% in younger adults, respectively. None of the participants took prophylactic paracetamol or nonsteroidal anti-inflammatory drugs. Unsolicited adverse events reported over the 50-day study period mainly consisted of cases of is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. rates in all ages were 93·8%, 100% and 100% for the 3 μg , 9 μg and 30 μg dose levels All groups had GMTs that were higher than those observed in convalescent sera; 144 (95% CI: 54-386], n = 21), and these remained higher than convalescent sera at Day 50. Following SCB-2019+CpG/Alum there were dose-dependent responses in young adults which were higher than in the older adults, and only matched the levels in convalescent sera with 9 μg and 30 μg doses of SCB-2019+CpG/Alum in the young adult groups, who had seroconversion rates of 50%, 80% and 93·3% after two doses (table 2) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.20243709 doi: medRxiv preprint 2019+AS03, 16 of 48 (33%) recipients across doses had seroconverted, increasing to 47 of 48 (98%) by Day 36, after the second dose. This increase in neutralising antibodies was SCB-2019 dose-dependent, shown by the geometric means of 1280, 1810 and 3948 MN50 in the 3 μg, 9 μg and 30 μg groups, respectively. Importantly the range of MN50 GMTs seen in older adult groups (1076-3320) were similar to those in the young adult groups for all dose levels in both age groups and were higher than convalescent sera (MN50 GMT 717; 95% CI 213-2417, n = 21). There was some decline in GMTs, but high levels of neutralising antibodies persisted to Day 50 in both age groups (Figure 5) . Dose-dependent increases in neutralising activity were also observed in the SCB-2019+CpG/Alum groups, but these responses were lower in magnitude than the AS03 groups as illustrated in figure 5 . In the older adult groups the range of MN50 GMTs (123-263) appeared lower than in the convalescent sera. High titres were maintained up to Day 50, the last timepoint tested in this interim analysis. When the correlations between immune responses assessed by the three different assays were investigated in convalescent and vaccinee sera there were highly significant linear relationships between each of the three assays (see Supplementary appendix pages 6-7). The is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.20243709 doi: medRxiv preprint The primary objective of this study was to assess the safety and reactogenicity of SCB-2019 when administered alone or as one of two adjuvanted formulations with AS03 or CpG and after the first dose, incidence rates of solicited adverse events were similar in both age groups after the second dose. The use of AS03 in pandemic H5N1 influenza vaccines allowed a demonstration of its general safety [15] , while large trials of the same vaccine revealed a higher local reactogenicity than we observed with injection pain in 89% of 18-64 year-olds [16] . Overall, this reactogenicity profile compares favourably with those of the mRNA SARS-CoV-2 vaccines which had incidence rates of local pain rates approaching or reaching 100% in adults [17, 18] . The rates of solicited AEs in the CpG/Alum-adjuvanted vaccine groups were lower and consistent with licensed CpG-adjuvanted vaccine [19, 20] . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.20243709 doi: medRxiv preprint observed with AS03 and after completion of the two dose series GMTs rapidly peaked at Day 36 at levels that were higher than those observed in convalescent sera from patients hospitalised with COVID-19 and the NIBSC standard sample. These high levels persisted until the end of this interim analysis as Day 50. There was little meaningful difference between the immune responses to SCB-2019+AS03 between the young and older adults. When adjuvanted with CpG and Alum, the immune responses were lower than with AS03 and SCB-2019 dose-dependent. Further, the response to SCB-2019+CpG/Alum were lower in the older age group. Further investigation of the cellular immune responses showed increases in Th1-polarised responses after both first and second doses for both AS03adjuvanted and CpG/Alum-adjuvanted SCB-2019. CD4+ T-cell responses have been suggested to complement humoral antibody responses in overcoming SARS-CoV-2 infection [19] . As a strong correlation between the neutralising activity and ELISA IgG antibody responses to S protein and RBD site has been seen in convalescent sera from PCR-confirmed COVID-19 patients [21] we investigated these ratios in our assays. We confirmed strong correlations between neutralising activity and IgG measured in either the SCB-2019 or ACE2-receptor assays. This is an important aspect of the immune response as it has been suggested that low neutralising/binding antibody ratios could contribute to an increased risk of antibodyenhanced disease [22] . Both adjuvanted formulations have been shown to be protective in preclinical non-human primate and rodent animal models, but AS03 formulations appeared to induce superior humoral immunogenicity and we observed an apparent lack of age-effect on the response. Although the AS03 formulation had higher reactogenicity compared with CpG/Alum, the severity appeared to be lower than to the mRNA and some vector- is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. In conclusion we have demonstrated safety, good tolerability and high neutralising immune responses with a Th1-biased cellular immune response that supports the further development of both candidate vaccines including assessment of the protective efficacy. MD, BM, BH, IS, PL, PL, HHH and JL are full-time employees and RC is a scientific advisor of the study sponsor, PR declares no conflict of interest. We are grateful to all the volunteers and the study staff at Nedlands, Western Australia. We wish to thank GSK and Dynavax for providing the AS03 and CpG 1018 adjuvants, respectively, and the Melbourne Health and VIDRL (Victorian Infectious Diseases Reference Laboratory) for providing a sample of SARS-CoV-2 for use in the virus neutralisation assays. The authors are grateful to Professor Jim Buttery and the SPEAC, and the Scientific Advisory is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Teich, Anh Wartel, and Nicholas Jackson) for helpful expert advice and support. We thank Keith Veitch (keithveitch communications, Amsterdam, the Netherlands) for drafting and editorial management of the manuscript. (CEPI). Any opinions, findings, and conclusions expressed in this material are those of the authors. The datasets, including the redacted study protocol, redacted statistical analysis plan, and individual participants data supporting the results reported in this article, will be available three months from initial request, to researchers who provide a methodologically sound proposal. The data will be provided after its de-identification, in compliance with applicable privacy laws, data protection and requirements for consent and anonymisation. MD, JL, RC, PR, BM, BH, PL, IS and HHH designed the study, data collection was by LH, and data analysis support by PL. Interpretation and writing of the manuscript was by all authors led by RC and a medical writer. All authors approved the submission for publication. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.20243709 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.20243709 doi: medRxiv preprint An SAE is any adverse event that: • Results in death is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.20243709 doi: medRxiv preprint Some interference with activity 3 Significant, prevents daily activity 4 ER visit or hospitalisation. . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.20243709 doi: medRxiv preprint Maxisorp plates were coated with 1μg/ml SCB-2019 at 4°C overnight and blocked with 2% non-fat milk in PBS containing 0·05% Tween-20 (PBST). Eight two-fold serial dilutions of serum samples starting from a 1:25 initial dilution were added to the blocked and washed SCB-2019-coated plates and incubated for 1h at 37°C. Plates were then washed, incubated with HRP-conjugated anti-human IgG for 1 hr at 37°C, washed again and the colorimetric signals were developed using TMB substrate for 3 min before stopping the reaction with 1N sulfuric acid. Optical density (OD) was measured at 450/650 nm. The EC50 of each test sample was calculated using a non-linear four parameter regression curve using GraphPad Prism, v.6.0c. Maxisorp plates were coated with 1 μg/mL ACE2-Fc at 4°C overnight, blocked with 2% nonfat milk in PBS containing 0·05% Tween-20 (PBST). On dilution plates equal volumes of eight two-fold, serial-diluted sera starting from a 1:5 initial dilution were incubated with Colorimetric signals were developed using TMB substrate until the OD650 of the positive control wells reached 0·7 when the reaction was stopped by addition of 1N sulfuric acid to the whole plate, and absorbance was promptly read at OD450/650nm. The 50% inhibition was calculated based on the negative and positive controls, and a non-linear four parameter regression curve was used to calculate the IC50 for each test sample with GraphPad Prism, v.6.0c. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.20243709 doi: medRxiv preprint Serum samples were heat inactivated for 30 minutes at 56⁰C and eleven two-fold serial dilutions of test samples were prepared in a separate dilution plate. Sera were mixed with an equal volume of SARS-CoV-2, hCoV-19/Australia/VIC01/2020 (GenBank MT007544.1), and incubated for 1 hr at 37°C, 5% CO2. The virus/serum mixtures (200 TCID50 units/well) were then transferred in duplicate to sub-confluent Vero E6 cell monolayer plates, preseeded 24 hours beforehand in 96 well plates at 1·5 x 10 4 cells/well. Plates were incubated for 3 days at 37°C, 5% CO2. The residual non-neutralised virus was detected via cytopathic effect (CPE) by microscopic scoring. The neutralisation titre is expressed as the reciprocal of the highest dilution at which 50% of the replicate wells were protected from infection (MN50). If at least 50% protection was not observed for an individual serum sample at any dilution, the MN50 titre was recorded as <20. . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.20243709 doi: medRxiv preprint Shown are the ratios of wild-type SARS-CoV-2 virus neutralisation titres (MN50) to SCB-2019 binding antibody IgG titres (EC50). Bars show geometric mean values for each group ± 95% confidence intervals (CI). Dots represent values from individual subjects. Yellow highlighted range represents min-max range (0·14-2·5) of ratios in convalescent sera (excluding NIBSC 20/130). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.20243709 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.20243709 doi: medRxiv preprint Global impact of COVID-19 infection requiring admission to the intensive care unit: a systematic review and meta-analysis WHO. 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