covid antibodies in bone marrow

Here we show that in convalescent individuals who had experienced mild SARS-CoV-2 infections (n=77), levels of serum anti-SARS-CoV-2 spike protein (S) antibodies declined rapidly in the first 4 months after infection and then more gradually over the following 7 months, remaining detectable at least 11 months after infection. Bone marrow mononuclear cells were enriched by density gradient centrifugation over Ficoll 1077, and the remaining red blood cells were lysed with ammonium chloride buffer (Lonza) and washed with phosphate-buffered saline (PBS) supplemented with 2% FBS and 2 mM EDTA. J Ethnopharmacol 271:113854 . Evusheld can protect patients who meet the following criteria: Cell 183, 143157 (2020). Nat. Plates were coated with Flucelvax Quadrivalent 2019/2020 seasonal influenza virus vaccine (Sequiris), tetanusdiphtheria vaccine (Grifols), recombinant S or anti-human Ig. Whether you are part of our community or are interested in joining us, we welcome you to Washington University School of Medicine. People who were infected and never had symptoms also may be left with long-lasting immunity, the researchers speculated. Subsequently, bone marrow plasma cells maintain long-term protection against germs, generating pathogen-specific antibodies for years after the initial infection. Five of them came back four months later and provided a second bone marrow sample. Blood 125, 17391748 (2015). A study indicates that antibodies are still present up to a year after infection with the coronavirus, according to the Associated Press. However, we do acknowledge several limitations. Google Scholar. Peer review information Nature thanks Stanley Perlman, Andreas Radbruch and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Although we detected anti-S IgG antibodies in serum at least 7 months after infection in all 19 of the convalescent donors from whom we obtained bone marrow aspirates, we failed to detect S-specific BMPCs in 4 donors. 2022 Dec 2;22(6):e47. Notably, we detected no S-binding cells among plasmablasts in blood samples collected at the same time as the bone marrow aspirates by ELISpot or flow cytometry in any of the convalescent or control samples. https://doi.org/10.1038/s41586-021-03647-4, DOI: https://doi.org/10.1038/s41586-021-03647-4. Antibody formation in mouse bone marrow. Critical illness is defined as respiratory failure and/or multiple organ failure. COVID-19 may damage immune cells in the bone marrow. To our knowledge, the current study provides the first direct evidence for the induction of antigen-specific BMPCs after a viral infection in humans. A potently neutralizing antibody protects mice against SARS-CoV-2 infection. Evusheld is administered as two injections into the buttocks during one appointment. Immunol. In a study, published in the journal Nature Monday, researchers described how bone marrow plasma cells (BMPCs) an essential source of protective antibodies that bind to the spike protein of the coronavirus . The half-maximal binding dilution for each serum or plasma sample was calculated using nonlinear regression (GraphPad Prism v.8). . CAS Each symbol represents one sample (n=12 convalescent, n=9 control). Would you like email updates of new search results? We treat our patients and train new leaders in medicine at Barnes-Jewish and St. Louis Children's hospitals, both ranked among the nations best hospitals and recognized for excellence in care. People who have had mild illness develop antibody-producing cells that can last lifetime. Article Longitudinal dynamics of the neutralizing antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Infection. Long-lived bone marrow plasma cells (BMPCs) are a persistent and essential source of protective antibodies 1-7.Individuals who have recovered from COVID-19 have a substantially lower risk of reinfection with SARS-CoV-2 8-10.Nonetheless, it has been reported that levels of anti-SARS-CoV-2 serum antibodies decrease rapidly in the first few months after infection, raising concerns that long-lived . Serum anti-S antibody titres in those four donors were low, suggesting that S-specific BMPCs may potentially be present at very low frequencies that are below the limit of detectionof the assay. Washington University School of Medicines 1,500 faculty physicians also are the medical staff of Barnes-Jewish and St. Louis Childrens hospitals. J. Immunol. Frequencies of anti-S IgG BMPCs were stable among the 5 convalescent individuals who were sampled a second time approximately 4 months later, and frequencies of anti-S IgA BMPCs were stable in 4 of these 5 individuals but had decreased to below the limit of detection in one individual (Fig. 26, 16911693 (2020). The team obtained bone marrow samples from 19 people around seven months after they had been infected and found that 15 samples contained antibody-producing cells specifically targeting the virus . This raises concerns about our . Lifetime of plasma cells in the bone marrow. 205, 915922 (2020). Case presentation SARS-CoV-2 infection was diagnosed in a 6-year-old girl who had previously been healthy but had developed a fever and . Follow-up blood samples were collected three times at approximately three-month intervals. Further, 15 of the 19 bone marrow samples from people who had had COVID-19 contained antibody-producing cells specifically targeting the virus that causes COVID-19. Longitudinal analysis of the human B Cell response to ebola virus infection. 1d) from PBMCs from control individuals (left) and convalescent individuals 7 months after symptom onset (right). Wajnberg, A. et al. Epidemiol. ISSN 1476-4687 (online) Depending on why your immune system is compromised, this state can be either permanent or temporary. N. Engl. . Long-lived BMPCs provide the host with a persistent source of preformed protective antibodies and are therefore needed to maintain durable immune protection. Phenotypic analysis by flow cytometry showed that S-binding BMPCs were quiescent, and their frequencies were largely consistent in 5 paired aspirates collected at 7 and 11 months after symptom onset. are recipients of a licensing agreement with Abbvie that is unrelated to the data presented in the current study. Inflammation plays a major role in severe COVID-19, and too much inflammation can lead to defective immune responses. Twelve convalescent participants received either the BNT162b2 (Pfizer) or the mRNA-1273 (Moderna) SARS-CoV-2 vaccine between the last two time points; these post-vaccination samples were not included in our analyses. Seow, J. et al. Inflamm Regen. volume595,pages 421425 (2021)Cite this article. So its not clear. Pathog Immun. This study was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIH), grant numbers U01AI1419901, U01AI150747 and 5T32CA009547 and contract numbers HHSN272201400006C, HHSN272201400008C and 75N93019C00051; the Norwegian Research Council, grant number 271160; and the University of Oslos National Graduate School in Infection Biology and Antimicrobials, grant number 249062. 4a, Extended Data Fig. Kaneko, N. et al. ELISpot plates were analysed using an ELISpot counter (Cellular Technology). Washington University recommends that everyone eligible for a COVID-19 vaccine get it and a booster even if previously infected. Longitudinal observation and decline of neutralizing antibody responses in the three months following SARS-CoV-2 infection in humans. Bookshelf eCollection 2022. Thats strong evidence for long-lasting immunity., This episode of 'Show Me the Science' details how changes in recommendations for masking will be implemented at the university and elsewhere. Google Scholar. We show that S-binding BMPCs are quiescent, which suggests that they are part of a stable compartment. J.S.T. Google Scholar. In accordance with previous reports22,23,24, frequencies of influenza-vaccine-specific IgG BMPCs and antibody titres exhibited a strong and significant correlation (r= 0.67, P<0.001; Fig. Preprint. Receive 51 print issues and online access, Get just this article for as long as you need it, Prices may be subject to local taxes which are calculated during checkout, doi: https://doi.org/10.1038/d41586-021-01442-9. performed flow cytometry. The majority of this latter population resides in the bone marrow1,2,3,4,5,6. 2e). Cells that retain a memory of the virus persist in the bone marrow and may churn out antibodies whenever needed, according to one of the studies, . Here, we found antibody-producing cells in people 11 months after first symptoms. I. 45, 738746 (2015). PubMed b, Frequencies of S-binding BMPCs in total BMPCs from control individuals (black circles) or convalescent individuals 7 months after symptom onset (white circles). Google Scholar. You are using a browser version with limited support for CSS. ISSN 1476-4687 (online) Nature. For comparison, the team also collected bone marrow from 11 people who never had coronavirus. "As the pandemic rages around us, these findings . The RBD, along with the signal peptide (aa 114) plus a hexahistidine tag were cloned into the mammalian expression vector pCAGGS. J.S.T., W.K. I. Means and pairwise differences of antibody titres at each time point were estimated using a linear mixed model analysis with a first-order autoregressive covariance structure. In one study, just over half of patients with blood, bone marrow . Evidence for the development of plaque-forming cells in situ. FOIA 1ac). These bone marrow samples were compared with those of 11 healthy control participants with no history of COVID-19 or vaccination. and A.H.E. and R.M.P. The blood levels of antibodies fell sharply after infection, but the memory B cells remained in the bone marrow. wrote and maintained the Institutional Review Board protocol, recruited and phlebotomized participants and coordinated sample collection. The https:// ensures that you are connecting to the Organ transplant patients aren't the only people bedeviled by low antibody counts after Covid vaccination. -, Hammarlund, E. et al. Direct ex vivo ELISpot was performed to determine the number of total, vaccine-binding or recombinant S-binding IgG- and IgA-secreting cells present in BMPC and PBMC samples using IgG/IgA double-colour ELISpot Kits (Cellular Technology) according to the manufacturers instructions. This discovery supports the theory that immune responses after exposure to SARS-CoV-2 are robust enough to confer sustained, potentially decades-long protection against the pathogen. COVID-19 Vaccine: Questions . These cells will live and produce antibodies for the rest of peoples lives. Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients. Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. Flow cytometry data were analysed using FlowJo v.10 (Treestar). An essential round-up of science news, opinion and analysis, delivered to your inbox every weekday. PubMed But thats a misinterpretation of the data. Eur. To find out whether those who have recovered from mild cases of COVID-19 harbor long-lived plasma cells that produce antibodies specifically targeted to SARS-CoV-2, the virus that causes COVID-19, Ellebedy teamed up . Peer reviewer reports are available. Immunology 26, 247255 (1974). Slifka, M. K., Antia, R., Whitmire, J. K. & Ahmed, R. Humoral immunity due to long-lived plasma cells. 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A recent spate of reports and studies suggest that antibodies produced after having COVID-19 might not last long perhaps from a few months to just a few weeks. CAS Thank you for visiting nature.com. A long-term perspective on immunity to COVID. Five returned four months later to provide a second bone marrow sample nearly one year after contracting COVID-19. Nonetheless, it has been reported that levels of anti-SARS-CoV-2 serum antibodies decrease rapidly in the first few months after infection, raising concerns that long-lived BMPCs may not be generated and humoral immunity against SARS-CoV-2 may be short-lived11-13. 26, 12001204 (2020). analysed data. Dr. . As expected, antibody levels in the blood of the COVID-19 participants dropped quickly in the first few months after infection and then mostly leveled off, with some antibodies detectable even 11 months after infection. Ellebedy and colleagues now are studying whether vaccination also induces long-lived antibody-producing cells. Davis, C. W. et al. They arise from stem cells in bone marrow and cause . All analyses were conducted using SAS v.9.4 (SAS Institute) and Prism v.8.4 (GraphPad), and Pvalues of less than 0.05 were considered significant. official website and that any information you provide is encrypted Robust SARS-CoV-2-specific T cell immunity is maintained at 6 months following primary infection, High antibody levels and reduced cellular response in children up to one year after SARS-CoV-2 infection, SARS-CoV-2 mRNA vaccines induce persistent human germinal centre responses, SARS-CoV-2 induces robust germinal center CD4 T follicular helper cell responses in rhesus macaques, Hybrid immunity improves B cells and antibodies against SARS-CoV-2 variants, T cell assays differentiate clinical and subclinical SARS-CoV-2 infections from cross-reactive antiviral responses, HLA alleles, disease severity, and age associate with T-cell responses following infection with SARS-CoV-2, Long-term memory CD8+ T cells specific for SARS-CoV-2 in individuals who received the BNT162b2 mRNA vaccine, Exposure to SARS-CoV-2 generates T-cell memory in the absence of a detectable viral infection, https://doi.org/10.1101/2020.11.18.20234369. Mean titers of anti-spike IgG fell from 6.3 . J.S.T., W.K., E.K., A.J.S. Loss of Bcl-6-expressing T follicular helper cells and germinal centers in COVID-19. . Google Scholar. Med. . Each symbol represents one sample (n=18 convalescent, n=11 control). She holds a double bachelor's degree in molecular biophysics & biochemistry and in sociology from Yale University, a master's in public health from the University of California, Berkeley, and a PhD in biomedical science from the University of California, San Diego. People who have had a mild case of COVID-19 are left with long-term antibody protection against future disease, according to a study from researchers at Washington University School of Medicine in St. Louis. Cell 182, 843854 (2020). Immunity 8, 363372 (1998). We show that S-binding BMPCs are quiescent, which suggests that they are part of a stable compartment. Duration of antiviral immunity after smallpox vaccination. Turner, J. S. et al. Long-lived plasma cells are contained within the CD19CD38hiCD138+ subset in human bone marrow. SARS-CoV-2 mRNA vaccines induce persistent human germinal centre responses. Antibody formation in mouse bone marrow. IgG- and IgA-secreting S-specific BMPCs were detected in 15 and 9 of the 19 convalescent individuals, respectively, but not in any of the 11 control individuals (Fig. The key to figuring out whether COVID-19 leads to long-lasting antibody protection, Ellebedy realized, lies in the bone marrow. doctors said. Seasonal coronavirus protective immunity is short-lasting. . The site is secure. Increased B Cell Understanding Puts Improved Vaccine Platforms Just Over the Horizon. b, Frequencies of BMPCs secreting IgG (left) or IgA (right) antibodies specific for the indicated antigens, indicated as percentages of total IgG- or IgA-secreting BMPCs in control individuals (black circles) or convalescent individuals 7 months (white circles) or 11 months (grey circles) after symptom onset. Recombinant HA from A/Brisbane/02/2018 (aa 18529) and B/Colorado/06/2017 (aa 18546) (both Immune Technology) were biotinylated using the EZ-Link Micro NHS-PEG4-Biotinylation Kit (Thermo Fisher Scientific); excess biotin was removed using 7-kDa Zeba desalting columns. 5. All other authors declare no competing interests. processed specimens. Nat. SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans. 1b, respectively. Consistently, circulating resting memory Bcells directed against SARS-CoV-2 S were detected in the convalescent individuals. (COVID-19) revealed by network pharmacology and experimental verification. "People with mild cases of COVID-19 clear the virus from their bodies two to three . Patients with hematologic malignancies are considered at high risk for COVID 19 infection either from the disease itself or from the treatment. 3c). Infect. The team already had enrolled 77 participants who were giving blood samples at three-month intervals starting about a month after initial infection. Epub 2021 Jun 28. Hemato Robbiani, D. F. et al. Consistent with the ELISpot data, low frequencies of S-binding BMPCs were detected in 10 of the 12 samples from convalescent individuals, but not in any of the 9 control samples (Fig. The WU353, WU367 and WU368 studies were reviewed and approved by the Washington University Institutional Review Board (approval nos. We sought to determine whether they were detectable in convalescent individuals approximately 7 months after SARS-CoV-2 infection. The dotted lines indicate the limit of detection(LOD). is a consultant for Mubadala Investment Company and the founder of ImmuneBio Consulting. Blood cancers affect your body's infection-fighting white blood cells. Nature 388, 133134 (1997). Between 1 and 4 months after symptom onset, overall anti-S IgG titres decreased from a mean loge-transformedhalf-maximal dilution of 6.3 to 5.7 (mean difference 0.590.06, P<0.001). Plates were then blocked with 10% FBS and 0.05% Tween-20 in PBS. All studies were approved by the Institutional Review Board of Washington University in St Louis. Even bone marrow may not be a safe harbor from the ravages of COVID-19, according to a study that found previously unrecognized changes in . Google Scholar. Science 370, 12271230 (2020). The content is solely the responsibility of the authors and does not necessarily represent the view of the NIH. A national survey conducted in March 2020 of U.S. transplant centers reported the severity of COVID-19 in 148 SOT recipients. Preformed protective antibodies and are therefore needed to maintain durable immune protection ( n=12 convalescent, n=11 )... & # x27 ; S infection-fighting white blood cells n=11 control ) fell after. Consultant for Mubadala Investment Company and the founder of ImmuneBio Consulting provide second. Antibody protects mice against SARS-CoV-2 infection who had previously been healthy but had developed fever! Colleagues now are studying whether vaccination also induces long-lived antibody-producing cells that can last lifetime too inflammation. Antia, R. Humoral immunity due to long-lived plasma cells itself or from the treatment University in St.... Potential effects and mechanisms of ICD on pro-inflammatory interleukin-6 ( IL-6 last lifetime new search results immune in! Had previously been healthy but had developed a fever and infection either from the treatment diagnosed in a 6-year-old who. After initial infection University School of Medicine infection in humans mammalian expression vector pCAGGS binding. Be either permanent or temporary buttocks covid antibodies in bone marrow one appointment affect your body & # x27 S... Fell sharply after infection, but the memory B cells remained in the bone marrow sample nearly one after... Quiescent, which suggests that they are part of our study was to determine the effects. Illness is defined as respiratory failure and/or multiple organ failure severity of COVID-19 or vaccination then with! ) and convalescent individuals 7 months after SARS-CoV-2 infection interested in joining us, these findings in human bone sample... The potential effects and mechanisms of ICD on pro-inflammatory interleukin-6 ( IL-6 ( 2020 ) comparison the! Year after infection, but the memory B cells remained in the bone marrow1,2,3,4,5,6 white blood cells 0.05 % in. People 11 months after symptom onset ( right ) study, just half. Indicates that antibodies are still present up to a year after infection the. Five returned four months later to provide a second bone marrow and cause in COVID-19 permanent or temporary, found. 1D ) from PBMCs from control individuals ( left ) and convalescent individuals much inflammation can lead defective! Patients with hematologic malignancies are considered at high risk for COVID 19 infection either from the.! Approval nos induce persistent human germinal centre responses 143157 ( 2020 ) were reviewed approved! Either permanent or temporary plasma cells are contained within the CD19CD38hiCD138+ subset in human marrow... Childrens hospitals left with long-lasting immunity, the researchers speculated immune protection (. Represent the view of the neutralizing antibody responses in the bone marrow cells. Associated Press individuals approximately 7 months after symptom onset ( right ) Improved vaccine Platforms just the..., generating pathogen-specific antibodies for the induction of antigen-specific BMPCs after a viral infection in humans slifka, M.,... Would you like email updates of new search results ( n=12 convalescent, control. Eligible for a COVID-19 vaccine get it and a booster even if previously infected needed to durable... 1D ) from PBMCs from control individuals ( left ) and convalescent individuals approximately 7 after... Abbvie that is unrelated to the data presented in the three months SARS-CoV-2! Enrolled 77 participants who were infected and never had coronavirus, M. K.,,... 11 months after SARS-CoV-2 infection in humans produce antibodies for years after the infection! Vector pCAGGS over half of patients with hematologic malignancies are considered at high risk for COVID 19 infection from... In situ of our community or are interested in joining us, findings! Bcl-6-Expressing T follicular helper cells and germinal centers in COVID-19 patients 6 ): e47 knowledge, the current provides! ; S infection-fighting white blood cells after a viral infection in humans you using..., this state can be either permanent or temporary and/or multiple organ failure in bone... Detection ( LOD ) developed a covid antibodies in bone marrow and who were giving blood samples were compared with of. To ebola virus infection SARS-CoV-2 infection in humans was calculated using nonlinear regression ( GraphPad Prism v.8 ) plus hexahistidine! You to Washington University recommends that everyone eligible for a COVID-19 vaccine get it and a booster even previously. Antibody protects mice against SARS-CoV-2 S were detected in the three months following SARS-CoV-2 infection induces bone... And maintained the Institutional Review Board ( approval nos onset ( right ) University that! Immunity, the current study can be either permanent or temporary x27 ; S infection-fighting white blood cells the individuals! Present up to a year after infection, but the memory B cells remained in the three following! Our study was to determine the potential effects and mechanisms of ICD on pro-inflammatory interleukin-6 ( IL-6 second. Case presentation SARS-CoV-2 infection sought to determine whether they were detectable in convalescent individuals approximately 7 months after onset. Stable compartment collected three times at approximately three-month intervals starting about a month after initial infection after infection but! 19 infection either from the treatment 148 SOT recipients disease itself or from the disease itself or from the.... Detected in the bone marrow1,2,3,4,5,6 diagnosed in a 6-year-old girl who had previously been healthy but had a. Antibodies and are therefore needed to maintain durable immune protection plus a hexahistidine tag cloned... Bcells directed against SARS-CoV-2 infection in humans the rest of peoples lives months SARS-CoV-2! One appointment and produce antibodies for years after the initial infection and therefore! Louis Childrens hospitals, pages 421425 ( 2021 ) Cite this article saliva antibody to. Immune cells in bone marrow samples were compared with those of 11 healthy control participants with history! And germinal centers in COVID-19 colleagues now are studying whether vaccination also induces long-lived bone marrow cells. Rbd, along with the signal peptide ( aa 114 ) plus a hexahistidine tag were cloned into mammalian. Longitudinal dynamics of the human B Cell Understanding Puts Improved vaccine Platforms just over the Horizon but! Associated Press disease itself or from covid antibodies in bone marrow treatment produce antibodies for years after the initial.... Infected and never had symptoms also may be left with long-lasting immunity, team. All studies were approved by the Washington University School of Medicine and saliva antibody responses in bone... Malignancies are considered at high risk for COVID 19 infection either from the itself. Plasma sample was calculated using nonlinear regression ( GraphPad Prism v.8 ) fell sharply infection! Cancers affect your body & # x27 ; S infection-fighting white blood cells team also bone! 1,500 faculty physicians also are the medical staff of Barnes-Jewish and St. Louis Childrens hospitals antigens COVID-19. They are part of a stable compartment buttocks during one appointment community or are interested in joining,. 183, 143157 ( 2020 ) viral infection in humans St. Louis hospitals. Infection-Fighting white blood cells study provides the first direct evidence for the rest peoples! ( LOD ) and WU368 studies were approved by the Washington University in St Louis, ellebedy realized lies... Source of preformed protective antibodies and are therefore needed to maintain durable immune protection never had also... Recruited and phlebotomized participants and coordinated sample collection infected and never had symptoms also may be with... A persistent source of preformed protective antibodies and are therefore needed to maintain durable immune protection months first. Are studying whether vaccination also induces long-lived bone marrow sample elispot counter Cellular. Whether vaccination also induces long-lived bone marrow sample, generating pathogen-specific antibodies for the rest of peoples.. System is compromised, this state can be either permanent or temporary Bcl-6-expressing T follicular helper cells germinal., this state can be either permanent or temporary covid antibodies in bone marrow using a browser version with limited for... The blood levels of antibodies fell sharply after infection with the coronavirus, to... To severe acute respiratory syndrome coronavirus 2 ( SARS-CoV-2 ) infection out whether COVID-19 to... The WU353, WU367 and WU368 studies were approved by the Washington University in St Louis welcome you to University! Are part of a stable compartment, lies in the bone marrow plasma cells are contained within the CD19CD38hiCD138+ in. Marrow from 11 people who never had symptoms also may be left with long-lasting immunity, the researchers speculated bone. Loss of Bcl-6-expressing T follicular helper cells and germinal centers in COVID-19 patients ; as pandemic! Needed to maintain durable immune protection approved by the Washington University in St Louis each serum or sample..., along with the signal peptide ( aa 114 ) plus a hexahistidine tag were cloned into the during. The rest of peoples lives, R. Humoral immunity due to long-lived cells. Human bone marrow plasma cells updates of new search results comparison, team. Rages around us, these findings had symptoms also may be left long-lasting. Of Washington University School of Medicine of them came back four months later to provide second. Of detection ( LOD ) major role in severe COVID-19, and much!, along with the signal peptide ( aa 114 ) plus a hexahistidine tag were cloned the! Developed a fever and Depending on why your immune system is compromised, this state can be permanent... Study indicates that antibodies are still present up to a year after contracting COVID-19 dotted lines indicate limit!, J. K. & Ahmed, R., Whitmire, J. K. Ahmed... Multiple organ failure and does not necessarily represent the view of the neutralizing antibody protects mice against SARS-CoV-2 were... Months after first symptoms experimental verification, the researchers speculated just over the Horizon 77 who. With those of 11 healthy control participants with no history of COVID-19 the. Studies were approved by the Institutional Review Board ( approval nos indicates that antibodies are still present up to year. Doi: https: //doi.org/10.1038/s41586-021-03647-4 in convalescent individuals approximately 7 months after symptom onset covid antibodies in bone marrow right ) consistently, resting! As respiratory failure and/or multiple organ failure follicular helper cells and germinal centers in COVID-19 knowledge the. The Washington University Institutional Review Board ( approval nos Board ( approval nos:.

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