S-specific IgG measurement was performed employing indirect ELISA as described previously56,67. When considering specific optimal cutoffs, agreement between each antibody binding assay and Genscript sVNT increased consistently from 0.03 units for the Siemens assay to 0.25 units for the Beckman assay (kappa = 0.79 and 0.77, respectively). First bivalent COVID-19 booster vaccine approved by UK medicines regulator). Nanomaterial Delivery Systems for mRNA Vaccines. The possible explanation of the higher detectable viral RNA found in 10 g compared to 1 g immunized mice (Fig. Since the outbreak of COVID-19, the world has raced to understand and accurately diagnose infection caused by SARS-CoV-2. Peletta, A. et al. Chutitorn Ketloy. In the present study, researchers quantified the neurological phenotypes induced in neurons by the SARS-CoV-2 S protein. 2c). It also markedly reduced viral RNA burden in serum and tissues. Jackson, L. A. et al. %PDF-1.7 sample is stable for 7 days at 2-8C once separated from a clot or red blood cells, or in a gel separator tube. In brief, mouse splenocytes at 5105 cells/well were cultured with SARS-CoV-2 spike peptide pools spanning the entire sequence of spike protein, 25 peptides/pool (Mimotopes, Mulgrave, Victoria, Australia) at a final concentration of 2g/mL at 37C, 5% CO2 for 40h. Pools 15 and 610 corresponded to S1 and S2 regions of spike protein, respectively. This was concordant with the previous findings that Omicron subvariants could evade NAb induced by the first-generation or WT-virus-based vaccines46. For tissue samples, RNeasy Mini Kit (QIAGEN, Hilden, Germany) was used following manufacturer instructions. Goat-anti-human IgG, goat-anti-mouse IgG, or goat-anti-rabbit IgG antibodies (all were diluted 1:10,000) conjugated with horseradish peroxidase (HRP) were used as secondary antibodies (all were from KPL, MD, USA) and detected by chemiluminescence substrate (Immobilon western, Millipore, CA, USA) then exposed to an X-ray film. The 5-fold serially diluted mice sera were added in duplicate. Labcorp test details for SARS-CoV-2 Semi-Quantitative Total Antibody, Spike . Here, we describe the preclinical studies of ChulaCov19, a SARS-CoV-2 mRNA encoding prefusion-unstabilized ectodomain spike protein encapsulated in lipid nanoparticles (LNP). The number of copies of viral RNA per sample was derived from standard curves of serial dilutions of IVTs (5, 50, 5 102, 5 103, 5 104, 5 105 RNA copies number or genomic equivalent (GE)/reaction were included. SD; standard deviation. The new semi-quantitative testing service is the latest addition to the company's existing menu of COVID-19 qualitative IgG and IgG/IgM test services. Additional group (Experiment 3) immunized with 5g of ChulaCov19 was included for evaluation of NAb durability as measured at week 18 (15 weeks after received the 2nd dose) and the boosting effect of 3rd ChulaCov19 dose administered at week 20. T-cell responses to SARS-CoV-2 can be indirectly tested with antigen tests (such as Elispot) that tests for cytokines produced (i.e. Laurent Kbaier, Vaccines (Basel) 10, 613 (2022). Cells were then fixed with 4% paraformaldehyde for 30min at RT. The study identified the number of pulses per electrode as the most prominent characteristic that differentiated the spike protein-treated wells from the control wells. COVID-19 antibody testing - Mayo Clinic Detailed amino sequence was shown in Supplementary File1. The ChulaCov19 vaccine development program has exactly this goal, striving to address the current and future pandemics in LMICs54. The RT-qPCR data showed that both doses of vaccine prevented the expression of SARS-CoV-2 viremia at 5 or 6 days after viral inoculation. Absorbance at 450nm was determined with a spectrophotometer. Differences were considered significant at p<0.05 with exact p-values shown. This would allow for identification of the corresponding thresholds, using high-throughput binding antibody assays. Nature 586, 578582 (2020). The use of a specific threshold for decision-making regarding PreP in immunocompromised patients must be taken with cautions due to limitations, mainly based on the nature and type of assay used to measure the antibody. PDF Understanding Your Test Results What tests did you do? SARS-CoV-2 Spike Kunkalikar, Bhavana. IgG2a and IgG1 subclasses were also assessed to determine Th1 and Th2 responses, respectively. Four antibody binding assays were used for serological testing according to the instructions of the manufacturer. The second dose of ChulaCov19 strongly augmented the IgG antibody levels with an increase of 10-19 folds, p<0.01 for all dose ranges (Fig. The same dosage of approved vaccines were used with a dose of 5g ChulaCov19 (1/10 of the human dose used in Phase 2 Trial). The average body weight by group from week 5 to week 5+6 days was demonstrated in Fig. Protection of K18-hACE2 mice and ferrets against SARS-CoV-2 challenge by a single-dose mucosal immunization with a parainfluenza virus 5-based COVID-19 vaccine. This finding implied that ChulaCov19 is highly immunogenic against WT (Wuhan-Hu1) strain. Before administering S1 to neurons on day zero, a human monoclonal anti-S1 antibody was sampled and neutralized using the antibody. Moreover, ChAdOx1: AZD1222 that used unmodified S has been shown to induce high level of NAb and T cells responses even after a single immunization dose in two mouse strains38. When RT-qPCR was used, although viral RNA was still detected in some tissues, both dosages demonstrated a 99-100% reduction of viral RNA in tested tissues when compared to the control group. The ethics committee waived the need for formal written informed consent from patients, as this study was performed on clinical data retrieved from routine tests; thus, no patient was specifically included in this study. One-day-old Vero E6 cells were used for measuring the level of neutralizing antibodies by live-virus micro-neutralization (micro-VNT50). New crop of COVID-19 mRNA vaccines could be easier to store, cheaper to use: Science [updated 5 April 2022; cited 30 August 2022]. In negative control (group 3), 5 mice were immunized with PBS instead of ChulaCov19 using the same schedule. While neurological signs have been documented in some, not all, coronavirus disease 2019 (COVID-19) patients, the precise mechanism by which viruses affect neuronal cells is still unknown and, thus, a subject of investigation. This program is a strong foundation for the fight against the next pandemic by increasing preparedness to make mRNA vaccine widely and timely accessible for LMICs, including Thailand. SARS-CoV-2 is an enveloped positive-sense single-stranded RNA beta coronavirus with a 30 kb polycistronic genome that encodes non-structural proteins (ORF1a and ORF1b, that are processed into Nsp1-16) at the 5-end, and structural proteins (S, E, M and N), and several other accessory factors (ORF3a . Mean spike-specific IFN- positive T cells for 0.2, 1, 10 and 30g were 166, 429, 1913, and 1378 SFC/106 splenocytes, respectively. LMICs received these vaccines much later and in shorter supply, as evidenced by the most recent statistic (as of 31 August 2022) that in several African countries less than 30% of the population has received at least one vaccine dose20. After 2 doses of ChulaCov19 or phosphate-buffered saline (PBS, control group) with a 3-week interval, K18-hACE2 mice were tested for NAb kinetics against live SARS-CoV-2 strain hCoV-19/Hongkong/VM20001061/2020. Gilles Antoniotti, SARS-CoV-2 Surrogate Virus Neutralization Test (sVNT) Kit (cPass) was purchased from Genscript (Piscataway, USA). ];V^srE]DwCyTPre_fyG;Cb@*\d$ j '-|,!]jF/J9r\s%3! Peletta, A. et al. Among the recently approved vaccines, mRNA modality seems to be the most efficacious as it induces high levels of desired immune responses and protects from severe symptoms16,17. Although the currently available vaccines do not completely prevent infection, they are efficacious in reducing severe symptoms of infected individuals11. Agreement between antibody binding assays and Genscript sVNT positive and negative results according to the reference cutoff (264 BAU/ml). According to French regulations, the study was approved by the French ethics committee (Health Data Hub, approval number: F20211217094518). Bellamkonda, N. et al. Background To accurately interpret COVID-19 seroprevalence surveys, knowledge of serum-IgG responses to SARS-CoV-2 with a better understanding of patients who do not seroconvert, is imperative. Differences were considered significant at p<0.05 with exact p-values shown. World Health Organization. SARS-CoV-2 Antibodies (NCVIGG, NCVIGQ)[NCVIGB], The qualitative detection of anti-Nucleocapsid IgG (NCVIGG) and the quantitative detection of anti-Spike IgG (NCVIGQ) antibodies. Bars represent the GMTs and 95% CI for each group. INSTRUCTIONS FOR USE CoV2T In response to the COVID19 pandemic and in preparation for future pandemics, Thailand has funded this mRNA vaccine development program from preclinical to manufacturing and clinical development. No positive detection of viral RNA was present in the 10g group of animals analyzed by ISH. Three out of five mice reached euthanasia criteria on Day 5, and symptoms progressed for the remaining two mice which met the criteria on Day 6. The particles were re-characterized at 6- and 12-month after manufacture for stability assessment. p<0.05 and p<0.01 are indicated by * and **, respectively. Tight junction protein occludin is an internalization factor for SARS For example, the psVNT-50 against BA.1 in the CoronaVac-prime/ChulaCov19-boost group (psVNT-50 GMT=875) was significantly higher (p<0.01) than homologous CoronaVac (psVNT-50 GMT=5.1) and homologous AZD1222 (psVNT-50 GMT=2.7) groups. Bloomberg. Frdrique Retornaz, The authors acknowledge all the members of the Chula VRC for their input and support. Article Nat Commun 13, 4610 (2022). Front Immunol 11, 1673 (2020). In a heterologous prime-boost study, ChulaCov19 booster dose generates a 7-fold increase of NAb against Wuhan-Hu1 WT virus and also significantly increases NAb response against Omicron (BA.1 and BA.4/5) when compared to homologous CoronaVac or AZD1222 vaccination. The proprietary lipid and LNP composition are described in patent application WO2020097540A161,62. Freyn, A. W. et al. Infect Dis Poverty 11, 53 (2022). An RBD virus-like particle vaccine for SARS-CoV-2 induces cross-variant CoronaVac induces lower neutralising activity against variants of concern than natural infection. RBD-VLP Vaccines Adjuvanted with Alum or SWE Protect K18-hACE2 Mice against SARS-CoV-2 VOC Challenge. Google Scholar. WW is an employee of BioNet-Asia, Co. Ltd. We have disclosed those interests fully to their affiliations, and we have in place an approved plan for managing any potential conflicts arising from licensing of the patents. Previous study by Eichinger KM, et al. Her academic background is in Pharmaceutical sciences and she holds a Bachelor's degree in Pharmacy. Quantitative SARS-CoV-2 anti-spike responses to Pfizer - PubMed Available from: https://www.bloomberg.com/graphics/covid-vaccine-tracker-global-distribution (2022). In addition, there was no anamnestic antibody response detected in the ChulaCov19 vaccinated mice after viral challenge (Fig. Vaccines (Basel) 9, (2021). Folegatti, P. M. et al. Cell 185, 24222433.e2413 (2022). Magnitude of asymptomatic COVID-19 cases throughout the course of infection: A systematic review and meta-analysis. Google Scholar. The test can provide information about how your body reacted to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Medicines and Healthcare products Regulatory Agency (2022). Data are presented as GMT of micro-VNT50 titer with 95% confident interval. Neurological phenotypes induced by SARS-CoV-2 spike protein in neurons. Tissues were collected at week 5+6 days for assessment of viral RNA. 1, 8 (2018). Overall, the rescue experiment provided compelling evidence that S1 was able to suppress burst activities when exposed to cells early in their developmental course. CAS Jairak, W. et al. Watanabe, Y. et al. : analysis and interpretation of results, M.G.A., K.T., P.K., N.Y., P.P., S.B., S.M., T.H., R.I.E., W.W., T.T., K.L., and J.H. This candidate vaccine has now completed non-clinical toxicity and biodistribution studies and has entered Phase 1 and 2 human trials. Figures were created with BioRender.com. A. Similar with the previous study, low level of viral RNA occasionally detected in survived mice was also reported by studies that used K18-hACE2 as a model28. https://www.news-medical.net/news/20230427/Neurological-phenotypes-induced-by-SARS-CoV-2-spike-protein-in-neurons.aspx. In the meantime, to ensure continued support, we are displaying the site without styles Adv. Google Scholar. https://ClinicalTrials.gov/show/NCT05605470 (2022). As expected, Omicron subvariants, especially BA.4/5, showed the largest drop in micro-VNT50 titers (Fig. Statistical significance was set at P < 0.05. At week 22, the psVNT-50 GMT for WT (Wuhan-Hu1), Delta (B.1.617.2), BA.1 and BA.4/5 were 25,539, 10,722, 2133, and 1707, respectively; 13-57 folds increase from the pre-boost baseline (Week18). xA 0 @L Laboratoire BioestrelBiogroup, Mouans-Sartoux, France, Affiliation: Slider with three articles shown per slide. Nat Immunol 21, 13271335 (2020). By clicking "Allow All" you agree to the storing of cookies on your device to enhance site navigation, Prolonged Protective Immunity Induced by Mild SARS-CoV-2 Infection of K18-hACE2 Mice. To obtain E.P., C.K., D.W., and K.R. Previous specific optimal cutoffs fitted perfectly to patients with incomplete vaccination: a perfect agreement was observed between Genscript sVNT and each antibody binding assays among these patients (results not shown). This test should not be used to diagnose or exclude acute SARS-CoV-2 infection. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. In this interview conducted at Pittcon 2023 in Philadelphia, Pennsylvania, we spoke to Ron Heeran, a speaker at the 2023 James L. Waters Symposium. Mice sera were further analyzed for NAb by psVNT50 test against the important recent VOCs, including Delta (B.1.617.2) variant and Omicron (BA.1 and BA.4/5) variants, and titers significantly decreased for all VOCs. Such unusual characteristics, in conjunction with a highly contagious profile, resulted in the rapid spreading of the virus worldwide. However, the slightly higher level compared to the 30-g group was not statistically significant. a Intracellular S protein expression examined by immunofluorescent assay employing anti-RBD, -S1, -S2 or PCS as primary antibody, the nuclei were counter stained with DAPI (blue). https://www.biorxiv.org/content/10.1101/2023.04.24.538161v1, Pregnant women show unique immune response to COVID-19, Study indicates that SARS-CoV-2 has evolved to gain increased replicative fitness and become well-adapted in epithelial cells of human airways, High-protein diet counters adaptive thermogenesis in prediabetic individuals. Funding: The author(s) received no specific funding for this work. 6b, c, Table1). A subgroup analysis was performed according to vaccination status (complete or incomplete). Quantitative data were reported using median and interquartile range (IQR), and qualitative data were reported using frequency and percentage. Experiment 1: dose-response of homologous ChulaCov19 prime/boost study, mice were immunized twice intramuscularly at 3 weeks interval of ChulaCov19 with dosage ranging from 0.2, 1, 10, to 30g. Bleeding was performed at 2 weeks following each dose (and at week 18 for Experiment 3). Sylvie Jordana, These results reflect that ChulaCov19 was highly immunogenic and induced a Th1-skewed response in mice. The team assessed the data using an algorithm devised in-house. The primary components of the SARS-CoV-2 structure are envelope (E), spike (S), membrane (M), and nucleocapsid (N) proteins. Walsh, E. E. et al. Molecular-based testing is used to diagnose COVID-19, and serologic testing of antibodies specific to SARS-CoV-2 is used to detect past infection. Available from: https://www.who.int/en/activities/tracking-SARS-CoV-2-variants (2022). Competing interests: The authors have declared that no competing interests exist. Then, HRP-conjugated secondary antibodies, including rabbit anti-mouse IgG, dilution 1:10,000 (KPL, MD, USA), -IgG1 (dilution 1:5000), or -IgG2a dilution 1:5000 (both were from Southern Biotech, AL, USA) were added for an additional 1h. After washing, the signals were detected by adding tetramethylbenzidine (TMB) substrate (BioLegend, San Diego, CA, USA). Interim Guidelines for COVID-19 Antibody Testing | CDC Zheng, J. et al. A vaccine efficacy of 80% was achieved with 264 binding antibody units (BAU)/ml (95% confidence interval [CI]: 108, 806) for anti-spike, and 506 BAU/ml (95% CI: 135, over limit) for anti- receptor-binding domain (RBD) antibodies. Baiersdorfer, M. et al. mRNA capping was performed by the trinucleotide cap1 analog, CleanCap (TriLink Biotechnologies, San Diego, CA, USA). Even though most COVID-19 patients are asymptomatic or only mildly symptomatic2,3,4, the virus is still eminently transmissible even during the early phases of the illness. mSphere 7, e0024322 (2022). This was consistent with the prior study in K18-hACE2 that intranasal inoculation with the similar range of virus caused death within 1 week22. World Health Organization. Recombinant S protein with abolished S1/S2 cleavage site was used as positive control in HEK293T-hACE-2 binding assay (right panel of 2b) and western blot (right lane of each panel in 2c). Anti-spike antibody response to natural SARS-CoV-2 infection in the All patients had received at least one dose of either Pfizer/BioNTech BNT162b2 or Moderna COVID-19 vaccine (Spikevax): 60 patients received Pfizer vaccine (87%) and 9 received Moderna vaccine (13%). Tian, J. H. et al. Fact Sheet for Healthcare Providers The purified mRNA-S (ChulaCov19) with undetectable endotoxin was tested for protein expression in VERO E6 cells. Available from: https://covid19.trackvaccines.org/agency/who (2022). There were few limitations in this study. Optimal cutoffs for distinguishing positivity were calculated using logistic regression on Genscript sVNT binary results (negative/positive), prior to the Youden index maximization approach on receiver operating characteristic curve results. On the basis of these data at present anti-SARS CoV-2 serological assays' results are not interchangeable, and, more importantly, individual immune monitoring should be performed with the same method. All samples were collected at the Alphabio Laboratory in Marseille, France (European Hospital, AlphabioBiogroup). Baseline NAb levels at week 0 of all mice were negative. Agreement between the antibody binding assays and the Genscript sVNT assay is shown in Table 2. A Single-Cycle Influenza A Virus-Based SARS-CoV-2 Vaccine Elicits Potent Immune Responses in a Mouse Model. The NAb titers were drastically enhanced after the second dose was given, p<0.01 for all dose ranges. For the Siemens assay, the optimal cutoff was within the same range as the reference cutoff (270 BAU/ml). A Facile Method for the Removal of dsRNA Contaminant from In Vitro-Transcribed mRNA. Nat Commun 11, 6013 (2020). The signal was amplified using a specific set of amplifiers (AMP1-6) as recommended by the manufacturer and was detected using a Fast Red solution for 5min at room temperature. K18-hACE2 transgenic mice are highly susceptible and displayed clinical signs following SARS-CoV-2 challenge22,23. EBioMedicine 77, 103902 (2022). Experiment 2: a prime/boost regimen of 5g of ChulaCov19 and 1/10 of human dosage of approved vaccines available during the study period, including viral-vectored (ChAdOx1; AZD1222, Lot A10062, Nonthaburi, Thailand) and inactivated (CoronaVac, Lot C202105081, Beijing, China) vaccines. Philippe Halfon, By Day 4 after challenge, two mice in PBS-receiving group (control) began to show clinical signs of anorexia, lethargy, and rough hair coat. Sera were collected at weeks 0, 2, 3, 4+6 days, and 5+6 days for NAb measurements. x2- p` ` \0`0e`X{StAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA^@%&D7(mylkv The data as well as the p values suggested that the anti-S1 antibody reversed the impact of S1 on bursting activities. 6c. As required by French regulations, patients attending clinical laboratories are informed that their biological results can be used for research purposes and that they are free to refuse to allow this (information annotated in the clinical laboratory report). The outcome strongly suggests that the RBD itself is sufficient to suppress surge activities. There is also a limitation regarding the two semi-quantitative antibody binding assays as a saturation limit could be reached because of their limited measurement range. Negative test results do not rule out the possibility of an infection with SARS-CoV-2. When considering a reference cutoff of 264 BAU/ml, the assays showed moderate to good agreement with Genscript sVNT, with strong variations of the kappa index from 0.52 for Beckman and Roche to 0.76 for Siemens (kappa = 0.72 for Abbott). Hence, the low micro-VNT50 titer in the homologous AZD1222 group might increase if the interval between each dose is longer than 4 weeks as used in this study. SARS-COV-2 Variants: Differences and Potential of Immune Evasion. Human codon-optimized sequences of the ectodomain of SARS-CoV-2 spike protein, amino acid position 1-1,210 (Wuhan Hu-1 complete genome, GenBank: MN908947.1, https://www.ncbi.nlm.nih.gov/nuccore/MN908947.1) was synthesized by GenScript, Piscataway, NJ, USA). The neurons were treated with similar S1 concentrations on day 12. Cells with approximately 8090% confluency were transfected with 1g of IVT ChulaCov19 using Lipofectamine MessengerMax (Invitrogen, Carlsbad, CA, USA) according to the manufacturer protocols. J Immunol 166, 16901697 (2001). Zheng, C. et al. Even as SARS-CoV-2 mutates, some human antibo | EurekAlert! All patients developed specific T cell responses by ELISpot and CoVITEST in time-points 2 and 3. 2b). Here we demonstrated that an LNP-encapsulated mRNA encoding a secreted form of prefusion nonstabilized ectodomain of SARS-CoV-2 spike protein ChulaCov19 was able to elicit robust, specific antibody and T-cell responses. In this episode of omg OMx, Bruker's science-driven podcast, Kate Stumpo interviews Daniel Hornburg, the VP of Proteomics at Seer, as they discuss the innovative technologies in plasma proteomics. Broad and timely access to effective vaccines in LMICs, particularly the most under-served settings, has always been limited during past pandemics and this has extended to COVID-1920. The study findings demonstrated a causal relationship between the SARS-CoV-2 S1 protein and in-vitro burst trends in neuronal populations, which can be reversed by antibody treatment. Stanislas Rebaudet, In contrast, undetectable fluorescent signals for S proteins were observed when HEK293T-hACE-2 were incubated with supernatant from untransfected cells (Fig. 200 0 obj <>]/Filter/FlateDecode/BitsPerComponent 8/Length 2211/Height 275>>stream Serologic Testing Serology testing measures the host antibody response in the form of immunoglobulins (Ig) such as IgM, IgA, or IgG following infection and/or vaccination. showed time-dependent changes in the comparability of different antibody tests with samples collected at different time points [26]. p<0.05 and p<0.01 are indicated by * and **, respectively. Int J Infect Dis 114, 252260 (2022). Slides were then incubated with protease plus for 20min at 40C in a HybEZTM oven (ACD) and subsequently incubated with the SARS-CoV-2 specific probe for 2h at 40C in the HybEZTM oven. Results are reported as AU/mL. The Euroimmun Anti-SARS-CoV-2 IgG and IgA tests are separate ELISAs that detect antibodies against the S1 subunit of the SARS-CoV-2 spike protein. 399, 924944 (2022). demonstrated that only Th2-dominant but not Th1/Th2 balanced response enhanced lung pathology in adjuvanted recombinant RSV immunized mice45. Copyright: 2023 Halfon et al. Per manufactures package insert protective level is 50.0 AU/mL. Emerg Infect Dis 27, 31783180 (2021). Tseng, C. T. et al. The score (0-5) was assigned according to the percent distribution of fluorescent-positive cells. This implies that ChulaCov19 could induce a long-lasting NAb, at least until 15 weeks postimmunization especially against WT (Wuhan-Hu1) and Delta (B.1.617.2) variants. Among the 1g group, only one tissue had very few positive cells, the nasal epithelium. Boosting with ChulaCov19, although not statistically significant, it could enhance the IFN- positive T cells by approximately 6.5 folds (p=0.1523) of the magnitude of T cells response in CoronaVac-primed mice (273 SFC/106 splenocytes). Furthermore, the immunity in immunocompromised individuals may be less robust than in healthy individuals and may wane more quickly. Cao, Y. et al. SARS-CoV-2 Testing | COVID-19 Treatment Guidelines This contrasts with SARS CoV-1 where peak viral shedding occurs after patients were already quite ill5,6. There was no detectable viremia in mice in both high or low-dose vaccine-treated groups while an average of 7.71104 GE/mL (ranged from 1.03103 3.75105 GE/mL) of viral RNA was detected in PBS-received mice, Fig. Immunogenicity and protective efficacy of SARS-CoV-2 mRNA vaccine encoding secreted non-stabilized spike in female mice, https://doi.org/10.1038/s41467-023-37795-0.