anti sars cov 2 spike protein test results interpretation anti sars cov 2 spike protein test results interpretation
These services aid in identifying a relative . Retrieved on May 01, 2023 from https://www.news-medical.net/news/20230427/Neurological-phenotypes-induced-by-SARS-CoV-2-spike-protein-in-neurons.aspx. Vaccination status was complete among 61 patients (88%). Front Immunol 11, 1673 (2020). SARS-CoV-2 is the name of the virus that causes coronavirus disease 2019 (COVID-19). ACS Cent Sci 7, 594602 (2021). The positive cut-off was the subtracted OD450+3SD. The ChAdOx1 vectored vaccine, AZD2816, induces strong immunogenicity against SARS-CoV-2 beta (B.1.351) and other variants of concern in preclinical studies. 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 results should always be assessed in conjunction with patient . Monovalent vs. bivalent vaccines Which is more effective against SARS-CoV-2? Guillaume Penaranda These medications are primarily indicated for individuals who are at high risk of severe illness or death from COVID-19, including those who are immunocompromised. Detection of antibodies to the SARS-CoV-2 spike glycoprotein in both serum and saliva enhances detection of infection A. Translating a Thin-Film Rehydration Method to Microfluidics for the Preparation of a SARS-CoV-2 DNA Vaccine: When Manufacturing Method Matters. For SARS-CoV-2, Wuhan lineage (Hong Kong/VM20001061/2020, NR-52282) used for micro-VNT50 that performed at AFRIMS was obtained through BEI Resources (NIAID, USA). Indeed, the BAU/ml values were performed only on the B.1.1.7 variant in neutralization assays and not on different strains of the virus; hence, there may be no relation between immune markers and disease outcome [7]. SARS-CoV-2 delta variant infection in domestic dogs and cats, Thailand. Stained cells were visualized under confocal microscope (ZEISS LSM 800, Carl Zeiss, Germany). In the latter VNT protocol, serum-virus mixtures were incubated in VERO E6 cells for 5 days. 4a). These tests should not be used to diagnosis or exclude acute SARS-CoV-2 infection. The 5-fold serially diluted mice sera were added in duplicate. Viral RNA was extracted from 140l serum and tissue samples using the QIAamp viral RNA mini kit (QIAGEN, Hilden, Germany). S0 was used to depict unprocessed S protein. Folegatti, P. M. et al. Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike. In the control group, 3 out of 5 mice reached euthanasia criteria on Day 5 hence only 2 mice were analyzed for body weight on Day 6 after challenge. Nosoconseil, Aix les Bains, France, * E-mail: guillaume.penaranda@biogroup.fr. Laboratoire AlphabioBiogroup, Marseille, France, The spike (S) protein of the virus, which contains the major neutralizing epitopes in the receptor binding domain (RBD) and N-terminal domain (NTD), has proven to be the most promising immunogen18. xA 0 @L Article The slides were dehydrated in 60C dry oven until completely dry and then dipped in Xylene before mounting with a mounting medium. The overall concordance between the antibody binding assays and the Genscript sVNT also increased consistently i.e., 11% increase for Roche (86% concordance), 10% increase for Beckman (88% concordance), 2% increase for Siemens (90% concordance), and 1% increase for the Abbott assay (88% concordance). Additional quality control to ensure the absence of double-stranded RNA (dsRNA) and endotoxin contamination prior to encapsulation into lipid nanoparticles (LNPs) were performed as described previously60. 199 0 obj <>stream Selective expansion of high- or low-avidity cytotoxic T lymphocytes and efficacy for adoptive immunotherapy. Vaccines (Basel) 10, 613 (2022). Alene, M. et al. Is there an association between COVID-19 and the risk of developing an autoimmune disease? Google Scholar. Beyond the techniques used for the viral detection, the inverse correlation between vaccine dosage and tissue viremia might be the results of the quality of T cell response induced by the high vaccine dosage. Thus, most recently approved vaccines employ full-length S (with or without modification) or whole virus (inactivated) as a target antigen19. Baiersdorfer, M. et al. In this interview conducted at Pittcon 2023 in Philadelphia, Pennsylvania, we spoke to Ron Heeran, a speaker at the 2023 James L. Waters Symposium. mRNA encapsulation was performed by Genevant Sciences Corporation (Vancouver, British Columbia, Canada). Competing interests: The authors have declared that no competing interests exist. SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 immunogenicity in baboons and protection in mice. Such unusual characteristics, in conjunction with a highly contagious profile, resulted in the rapid spreading of the virus worldwide. All p values <0.05 were defined as statistically significant. Limited and Short-Lasting Virus Neutralizing Titers Induced by Inactivated SARS-CoV-2 Vaccine. When correlating protective efficacy and NAb titers induced by ChulaCov19, a micro-VNT50 titer of 2,560 before challenge in 1 g immunized mice was found to completely prevent viral burden in the lung as analyzed by ISH and RT-qPCR (Figs. Statistical significance was determined by two-sided MannWhitney test. J Control Release 217, 345351 (2015). van Doremalen, N. et al. Internet Explorer). In this study, the S1 and S2 subunits of the spike protein . Voysey, M. et al. Figures were created with BioRender.com. 2b). Furthermore, the immunity in immunocompromised individuals may be less robust than in healthy individuals and may wane more quickly. Cevik, M. et al. COVID-19 CORONAVIRUS PANDEMIC [updated 19 August 2022; cited 2022 19 August 2022]. You should not interpret the results of this test as an indication or degree of immunity or Pardi, N. et al. The opinions expressed here are the views of the writer and do not necessarily reflect the views and opinions of News Medical. % 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. Testing for SARS-CoV-2 Infection. Blood was collected at wk0, wk2, wk3, wk4+6 and wk5+6 days for antibody kinetic analysis (Fig. They were widely available in these countries for approximately a year before being accessible on other continents. Using the Youden index maximization approach, optimal cutoffs were consistently lower than the reference cutoff of 264 BAU/ml for the Abbott and Beckman assays (148 and 48 BAU/ml, respectively). Therefore SARS-CoV-2 serology may be standardized. ISSN 2041-1723 (online). Her college project work based on The manifestations and causes of sickle cell anemia formed the stepping stone to a life-long fascination with human pathophysiology. One of these was the low number of samples that were subjected to antibody quantification and the absence of an independent international standard (WHO in IU/ml). 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. Chen, X. et al. Comparisons of the data between groups were made using non-parametric tests (MannWhitney test). The Abbott AdviseDx SARS-CoV-2 IgG II immunoassay detects antibodies to the viral spike protein (S). Med. 01 May 2023. Presently, the pandemic is still surging in many countries. Mol Ther Nucleic Acids 15, 2635 (2019). RA-MF-28/64. K18-hACE2 transgenic mice are highly susceptible and displayed clinical signs following SARS-CoV-2 challenge22,23. The proprietary lipid and LNP composition are described in patent application WO2020097540A161,62. Moreover, the tissue slides were examined unblind. Bars (a) or horizontal lines (b) represent the geometric mean (GMT) for each group while error bars indicate the 95% confident interval. Lv, J., Wu, H., Xu, J. Nature 584, 450456 (2020). Although several SARS-CoV-2 vaccines used an engineered S protein to abolish S1/S2 cleavage or to stabilize the prefusion stage35,36,37, vaccines encoding unmodified S protein are also worth exploring as its structure is the same as native viral protein. 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). Cao, Y. et al. Secreted S protein was also subjected for analysis of its binding capability to hACE2. DNA Vaccine Administered by Cationic Lipoplexes or by In Vivo Electroporation Induces Comparable Antibody Responses against SARS-CoV-2 in Mice. Lipid nanoparticles). Of interest, the heterologous AZD1222-prime/ChulaCov19-boost induced the best specific T cells responses with mean spike-specific IFN- positive T cells of 3725 SFC/106 splenocytes, which approximately 1.7-fold higher than homologous ChulaCov19 (p=0.1934) and also significantly higher than other groups (p<0.05). Moreover, the low dose regimen was also shown to induce a marked reduction in viral load in nasal turbinates, brain, and lung tissues compared to sham-treated controls. In mice, ChulaCov19 was highly immunogenic as a booster in settings primed with either inactivated or viral vector vaccine. The study suggested that S1 is responsible for decreasing burst activities of neuronal populations when cells are exposed early in the course of development. ChAdOx1 nCoV-19 (AZD1222) or nCoV-19-Beta (AZD2816) protect Syrian hamsters against Beta Delta and Omicron variants. endstream Current SARS-CoV-2 antibody tests detect IgM or IgG to viral spike or nucleocapsid proteins. Available from: https://www.worldometers.info/coronavirus (2022). COVID-19 antibody testing is a blood test. Jairak, W. et al. 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). Protective activity of mRNA vaccines against ancestral and variant SARS-CoV-2 strains. the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in The S protein facilitates virus attachment and entrance into the host cell. 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. The test can provide information about how your body reacted to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Together with the emergence of new VOCs, a booster dose (either homologous or heterologous vaccine modality) is required to enhance the vaccine effectiveness15. Within the brain, ACE2 is predominantly expressed in the brain stem and regions whose primary function is to regulate blood pressure and cardiovascular function. S-specific total IgG analyzed at week 2 revealed that all ChulaCov19-immunized mice, either with 1 or 2 doses, elicited anti-S-specific IgG response from the lowest dose of 0.2g with a dose-dependent response pattern. At week 5 (2 weeks after the second dose), all mice in both vaccinated groups showed increased NAb levels. SARS-CoV-2 RNA-positive cells were examined and counted unblind by certified personnel. In contrast, a higher dose vaccination not only induced the mixture of low and high avidity T cells responses, but also induced the clonal deletion of high avidity CD8 T cells29,30,31. 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. Nat Commun 13, 4610 (2022). As the Omicron subvariant BA.4/5 is currently spreading worldwide, we have also assessed cross-neutralization and found that the NAb GMT measured by psVNT50 against BA.4/5 in homologous ChulaCov19 vaccination or heterologous boosted with ChulaCov19 groups were significantly better than either of the CoronaVac or AZD1222 homologous vaccination (Fig. 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. A. Its worth to mention that, as of now, theres no widely accepted cutoff value for immunity in immunocompromised patients, but some studies have suggested that antibody levels cut off may be associated with protection against COVID-19. The results revealed that the NAb against WT (Wuhan-Hu1) and Delta (B.1.617.2) variants were still detectable in all mice (5/5) but 4/5 mice for Omicron BA.1 and BA.4/5. Article Statistical significance was set at P < 0.05. Proc Natl Acad Sci U S A 114, E7348E7357 (2017). Differences were considered significant at p<0.05 with exact p-values shown. Jackson, L. A. et al. Adv. By Day 4 after challenge, two mice in PBS-receiving group (control) began to show clinical signs of anorexia, lethargy, and rough hair coat. Unfortunately, it has also been proven that vaccine efficacy decreases over time14. In brief, mouse splenocytes at 510 5 cells/well were cultured with SARS-CoV-2 spike peptide pools spanning the entire sequence of spike protein, 25 peptides/pool (Mimotopes, Mulgrave, Victoria . Correspondence to The purified mRNA-S (ChulaCov19) with undetectable endotoxin was tested for protein expression in VERO E6 cells. Of note, at week 5, all vaccinated mice at the 10 g dose, and 5 of 6 mice at 1 g dose elicited SARS-CoV-2 specific serum IgA (supplementary FigureS1a and S1b). Laboratoires Oriade NovialeBiogroup, Grenoble, France, Affiliation: 4c). Ma, Q. et al. Agreements between antibody-binding assays and Genscript sVNT were performed using Cohens kappa, crude concordance rate, and area under curve (AUC). 4d). The GE per gram of virus in a tissue sample was calculated by multiplying the number of copies/reaction by [10,000 x the weight of a tissue sample (mg) used for extraction]. : 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. Moreover, the feasibility of large-scale production as well as rapid adaptability to new variants are major advantages of the mRNA production platform. PubMed Central 4b). At this time-point, the NAb titers against both Omicron subvariants were still in the same level with week 5 titers (Fig. Lancet 396, 467478 (2020). This observation correlates with that of a recent clinical study report53. Recommendations based on only one study is not prudent. In addition, there was no anamnestic antibody response detected in the ChulaCov19 vaccinated mice after viral challenge (Fig. The NT50 titers against WT and Delta variants increased 7- to 14-fold when using the heterologous approach with ChulaCov19 as compared to the homologous immunizations with CoronaVac or AZD1222 (Fig. No serologic tests for SARS-CoV-2 are approved by the FDA; some, but not all, commercially available serologic tests for SARS-CoV-2 have received EUAs . Methods Protoc. Therefore, we suggest specific BAU/ml adjusted thresholds for the four commercial antibody assays (Abbott, Beckman, Roche, and Siemens), which can be used to guide the use of PreP in immunocompromised patients. In conclusion, whether it is generally believed that a certain level of antibodies is necessary to confer protection against the virus, but the exact level required is not yet clear. ChulaCov19 was further compared to two approved vaccines (CoronaVac and AZD1222), either in a homologous prime/boost setting or heterologous one (i.e. Available from: https://www.science.org/content/article/new-crop-covid-19-mrna-vaccines-could-be-easier-store-cheaper-use (2022). The female/male ratio was 67/33, and the median age was 47 years (IQR 3463). Vero E6 and HEK293T-hACE-2 were grown in Eagles minimum essential medium (EMEM) and Dulbeccos Modified Eagles Medium (DMEM), respectively supplemented with 5-10% heat-inactivated fetal bovine serum (HIFBS), 1% L-glutamine, 1% Pen/Strep, 40g/ml gentamicin and 0.25g/ml fungizone (all were from Invitrogen, Carlsbad, CA, USA) at 352 oC with 5% CO2. Quantitative data were reported using median and interquartile range (IQR), and qualitative data were reported using frequency and percentage. Neurological phenotypes induced by SARS-CoV-2 spike protein in neurons. This study was performed using sera collected between October 2021 and December 2021 from a real life cohort of 69 individuals attending internal medicine and infectious diseases department of the European Hospital (Marseille). Google Scholar. N Engl J Med 383, 19201931 (2020). Please note that medical information found At 2 weeks after the second immunization, mice were challenged intranasally with 2104 pfu (in 50L) of SARS-CoV-2 (wild-type). Vero E6, green monkey kidney epithelial cell line, was obtained from ATCC (Old Town Manassas, VA, USA). The promising preclinical study results presented here demonstrate that ChulaCov19 is highly immunogenic with protective efficacy. Available from: https://www.bloomberg.com/graphics/covid-vaccine-tracker-global-distribution (2022). The NT50 titer decrease found in our study was similar to those of other approved vaccines as the titers against BA.1 and BA.4/5 decreased by more than 8-10 folds when compared to the WT virus46,47,48. As expected, Omicron subvariants, especially BA.4/5, showed the largest drop in micro-VNT50 titers (Fig. The average body weight by group from week 5 to week 5+6 days was demonstrated in Fig. 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. This study was performed on data retrieved from 69 individuals, who received at least one dose of the Pfizer/BioNTech BNT162b2 or Moderna COVID-19 vaccine (Spikevax) at the Alphabio Laboratory in Marseille, France (European Hospital, AlphabioBiogroup). Sci. Animals were immunized IM with 1g or 10g of ChulaCov19 at weeks 0 and 3. However, harmonization of neutralizing antibody titers is necessary to determine a common threshold using which vaccine protection can be predicted. 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. Mol Ther 28, 15691584 (2020). van Doremalen, N. et al. Detailed amino sequence was shown in Supplementary File1. SARS-CoV-2 RNA levels in serum and tissue samples were quantitated using quantitative RT-PCR. Here, we describe the construction and preclinical evaluation of mRNA expressing the ectodomain of native, prefusion-non-stabilized S protein of wild-type (WT) Wuhan-Hu1 strain encapsulated within lipid nanoparticles, henceforth referred to as ChulaCov19. 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. Freyn, A. W. et al. Cell nuclei were counter stained with 4, 6-diamino-2-phenylindole hydrochloride (DAPI) (Sigma-Aldrich, USA). PubMed NAb measurements in mice sera from Experiment 1 against WT (Wuhan-Hu1) live-virus (micro-VNT50) at 2-week after each dose showed NAb response in a dose-dependent manner. Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. RNA copies were calculated as genomic equivalent/mg of tissue. For patients who do not regularly seek care within UW Medicine, our phlebotomists at the University of Washington Medical Center-Northwest Campus (UWMC-NW) and UWMC-NW Outpatient Medical Center (OPMC) located on Meridian Ave. N. are able to perform blood draws for testing with a valid provider order. Zhang, N. N. et al. 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). Article Article EP was also supported by Faculty of Medicine, Chulalongkorn University, grant No. PubMed Central ~N6R}q71m*:H^J6n1i2bcPh)\R ^Zei%|kz1>85k7^T\Lt)h\=-an}mr^yU+HNGlcE'&ps4toE4/f_=3A>o'9p:4ZUI1s+QtSIwu,\\Uv+QG Pm2+T`2T\y/3^?8UlVshDEY/qo_F%3^>D?lq2g R61sbWP}O3,p?K|3Tx=RdR'_/KzVFS j\h1s1vdzV|:p Data Availability: All relevant data are within the manuscript and its Supporting Information files. COVID-19 treatments and pathogenesis including anosmia in K18-hACE2 mice. These common antibody tests use purified proteins of SARS-CoV-2 (not a live virus) to detect the presence of binding antibodies that attach to a virus, per the CDC. Tracking SARS-CoV-2 variants 2022 [updated 11 August 2022; cited 2022 19 August]. Emerg Infect Dis 27, 31783180 (2021). Developing mRNA vaccine technology for distribution in these regions is therefore extremely important21. %PDF-1.7 Experiment 2: heterologous prime-boost study, mice were primed with 1/10 of the approved human dosage of CoronaVac or AZD1222 and boosted 4 weeks later with 5g of ChulaCov19. 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. Watanabe, Y. et al. By clicking "Allow All" you agree to the storing of cookies on your device to enhance site navigation, The threshold or cutoff value for immunity in immunocompromised individuals in relation to COVID-19 is currently not well established. : study conception and design, E.P., K.T., and C.K. Meta-analysis shows phytosterol-fortified foods effectively lower LDL cholesterol levels. 200 0 obj <>]/Filter/FlateDecode/BitsPerComponent 8/Length 2211/Height 275>>stream Covid-19 Tracker: More Than 12.6 Billion Shots Given [updated 31 August 2022; cited 2022 31 August]. Spin within 24 hours and prior to shipment. In the detection step, staining of the living cells with 0.02% neutral red (Sigma Aldrich, USA) in 1X PBS (Invitrogen, Carlsbad, CA, USA) was used instead of viral protein staining employing anti-nucleocapsid (1:5,000) used in Experiment 1. 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). Source data are provided as a Source Data file. Bhavana Kunkalikar is a medical writer based in Goa, India. This assay detects antibodies that block the interaction of SARS-CoV-2 with its entry receptor angiotensin-converting enzyme 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). CAS Rotshild, V., Hirsh-Raccah, B., Miskin, I., Muszkat, M. & Matok, I. Lancet Infect Dis 21, 13521354 (2021). 2023. Cells with approximately 8090% confluency were transfected with 1g of IVT ChulaCov19 using Lipofectamine MessengerMax (Invitrogen, Carlsbad, CA, USA) according to the manufacturer protocols. Her academic background is in Pharmaceutical sciences and she holds a Bachelor's degree in Pharmacy. An adjuvanted subunit SARS-CoV-2 spike protein vaccine provides protection against Covid-19 infection and transmission, Immunogenicity and protection of a variant nanoparticle vaccine that confers broad neutralization against SARS-CoV-2 variants, CpG-adjuvanted stable prefusion SARS-CoV-2 spike protein protected hamsters from SARS-CoV-2 challenge, Protection of hamsters challenged with SARS-CoV-2 after two doses of MVC-COV1901 vaccine followed by a single intranasal booster with nanoemulsion adjuvanted S-2P vaccine, mRNA-based SARS-CoV-2 vaccine candidate CVnCoV induces high levels of virus-neutralising antibodies and mediates protection in rodents, Intranasal immunization with a proteosome-adjuvanted SARS-CoV-2 spike protein-based vaccine is immunogenic and efficacious in mice and hamsters, Booster vaccination with Ad26.COV2.S or an Omicron-adapted vaccine in pre-immune hamsters protects against Omicron BA.2, The SARS-CoV-2 spike residues 616/644 and 1138/1169 delineate two antibody epitopes in COVID-19 mRNA COMIRNATY vaccine (Pfizer/BioNTech), A core-shell structured COVID-19 mRNA vaccine with favorable biodistribution pattern and promising immunity, https://www.ncbi.nlm.nih.gov/nuccore/MN908947.1, https://www.who.int/en/activities/tracking-SARS-CoV-2-variants, https://www.worldometers.info/coronavirus, https://covid19.trackvaccines.org/agency/who, https://apps.who.int/iris/handle/10665/363344, https://www.bloomberg.com/graphics/covid-vaccine-tracker-global-distribution, https://www.who.int/initiatives/the-mrna-vaccine-technology-transfer-hub, https://www.science.org/content/article/new-crop-covid-19-mrna-vaccines-could-be-easier-store-cheaper-use, https://ClinicalTrials.gov/show/NCT05231369, https://ClinicalTrials.gov/show/NCT05605470, http://creativecommons.org/licenses/by/4.0/.
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