key: cord-0830423-bptgfxmu authors: Li, Maggie; Beck, Evan J.; Laeyendecker, Oliver; Eby, Yolanda; Tobian, Aaron AR; Caturegli, Patrizio; Wouters, Camille; Chiklis, Gregory R.; Block, William; McKie, Robert O.; Joyner, Michael J.; Wiltshire, Timothy D.; Dietz, Allan B.; Gniadek, Thomas J.; Shapiro, Arell J.; Yarava, Anusha; Lane, Karen; Hanley, Daniel F.; Bloch, Evan M.; Shoham, Shmuel; Cachay, Edward R.; Meisenberg, Barry R.; Huaman, Moises A.; Fukuta, Yuriko; Patel, Bela; Heath, Sonya L.; Levine, Adam C.; Paxton, James H.; Anjan, Shweta; Gerber, Jonathan M.; Gebo, Kelly A.; Casadevall, Arturo; Pekosz, Andrew; Sullivan, David J. title: Convalescent plasma with a high level of virus-specific antibody effectively neutralizes SARS-CoV-2 variants of concern date: 2022-04-21 journal: Blood Adv DOI: 10.1182/bloodadvances.2022007410 sha: 8a8a2a29e435842ed121a941b9237077567f2055 doc_id: 830423 cord_uid: bptgfxmu The ongoing evolution of SARS-Co-V2 variants to omicron severely limits available effective monoclonal antibody therapies. Effective drugs are also supply limited. Covid-19 convalescent plasma (CCP) qualified for high antibody levels effectively reduces immunocompetent outpatient hospitalization. The FDA currently allows outpatient CCP for the immunosuppressed. Viral specific antibody levels in CCP can range ten- to hundred-fold between donors unlike the uniform viral specific monoclonal antibody dosing. Limited data are available on the efficacy of polyclonal CCP to neutralize variants. We examined 108 pre-delta/pre-omicron donor units obtained before March 2021, 20 post-delta COVID-19/post-vaccination units and one pre-delta/pre-omicron hyperimmunoglobulin preparation for variant specific virus (vaccine-related isolate (WA-1), delta and omicron) neutralization correlated to Euroimmun S1 IgG antibody levels. We observed a 2- to 4-fold and 20- to 40-fold drop in virus neutralization from SARS-CoV-2 WA-1 to delta or omicron, respectively. CCP antibody levels in the upper 10% of the 108 donations as well as 100% of the post-delta COVID-19/post-vaccination units and the hyperimmunoglobulin effectively neutralized all three variants. High-titer CCP neutralizes SARS-CoV-2 variants despite no previous donor exposure to the variants. 1 Convalescent plasma with a high level of virus-specific antibody effectively neutralizes SARS-CoV-2 variants of concern Short title: Convalescent Plasma Neutralizes Variant SARS-CoV-2 Maggie Li 1 , Evan J. Beck 2 , Oliver Laeyendecker 2 , Yolanda Eby 3 , Aaron AR Tobian 3 , Patrizio The ongoing evolution of SARS-Co-V2 variants to omicron severely limits available effective monoclonal antibody therapies. Effective drugs are also supply limited. Covid-19 convalescent plasma (CCP) qualified for high antibody levels effectively reduces immunocompetent outpatient hospitalization. The FDA currently allows outpatient CCP for the immunosuppressed. Viral specific antibody levels in CCP can range ten-to hundred-fold between donors unlike the uniform viral specific monoclonal antibody dosing. Limited data are available on the efficacy of polyclonal CCP to neutralize variants. We examined 108 predelta/pre-omicron donor units obtained before March 2021, 20 post-delta COVID-19/postvaccination units and one pre-delta/pre-omicron hyperimmunoglobulin preparation for variant specific virus (vaccine-related isolate (WA-1), delta and omicron) neutralization correlated to Euroimmun S1 IgG antibody levels. We observed a 2-to 4-fold and 20-to 40-fold drop in virus neutralization from SARS-CoV-2 WA-1 to delta or omicron, respectively. CCP antibody levels in the upper 10% of the 108 donations as well as 100% of the post-delta COVID-19/postvaccination units and the hyperimmunoglobulin effectively neutralized all three variants. Hightiter CCP neutralizes SARS-CoV-2 variants despite no previous donor exposure to the variants. Commercial serologic assays predictive of SARS-CoV-2 and variant neutralization are important for effective clinical use of COVID-19 convalescent plasma (CCP), because substantial heterogeneity exists in CCP donor responses with higher antibody levels associated with virus neutralization. [1] [2] [3] The SARS-CoV-2 omicron variant BA.1 rendered many monoclonals ineffective in laboratory virus neutralization tests, necessitating their removal as outpatient monoclonal therapies for acute 5 While Sotrovimab retained activity against omicron BA.1, activity was lost against the BA.1.1 and BA.2 Omicron variants. Tixagevimab/cilgavimab (Evusheld), approved only for post-exposure prophylaxis, was ineffective at neutralizing omicron BA.2 and showed reduced activity towards omicrons BA.1 and BA.1.1. 6 Bebtelovimab neutralizes omicron BA.1, but will have limited availability. 7 A recent large clinical trial over the period from June 2020 to October 2021 demonstrated that early outpatient CCP reduced the risk of hospitalizations by more than half. 8 Ninety percent of the trial's 300+ unique CCP donor units were collected prior to January, 2021 representing nonvaccinated pre-alpha/delta/omicron variant plasma. In December 2021, the FDA extended CCP from hospital use to immunosuppressed outpatients, while simultaneously increasing commercial serologic benchmarks by 1.5-fold for CCP qualification. 9 Recent omicron variant studies measured virus neutralization without concomitant commercial serologic testing for general antibody levels. 5 Wang and colleagues measured a 10fold virus neutralization reduction from SARS-CoV-2 wild type to omicron in 16 individual plasma samples obtained from January to March 2020 in China. 10 Röllser et al examined only 10 CCP units from donors infected with variants and showed a lack of CCP neutralization of omicron, but substantial neutralization with post COVID-19 post vaccination plasma. 11 These studies have only tested a few samples and do not correlate the results to a commercial assay necessary to qualify therapeutic CCP units. Considering that CCP donors vary significantly in terms of virus neutralization capacity, the use of commercial assays that predict neutralization of SARS-CoV-2 and its variants becomes important to select the optimal therapeutic CCP units. This study tested a total of 129 samples: 108 CCP donor units, 20 post-delta COVID-19/post-vaccination donor units and one pre-delta/pre-omicron hyperimmunoglobulin preparation for ability to neutralize three SARS-CoV-2 isolates/variants (WA-1, delta, and omicron), correlating results to the Euroimmun spike-S1 antibody plasma level. After the outpatient clinical research trial transfusions from 300+ unique donors were complete, 108 remnant qualified (positive antibody presence after 1:320 dilution using a validated spike protein CLIA ELISA assay) donor plasma units were available for WA-1, delta, and omicron virus neutralizations. 8 The hyperimmunoglobulin sample was prepared from 101 CCP units, collected between June 16, 2020 and January 13, 2021 (representing pre-delta/pre-omicron plasma) from recovered patients by an apheresis plasma collection technique following Mayo Clinic Blood Donor Center's standard operating procedures. Pooled plasma was loaded on the protein A resin to capture IgG at a neutral pH, washed to remove low affinity proteins and eluted at low pH. Final product was formulated as a 5% protein solution in glycine/acetic acid. Plasma neutralizing antibodies (nAbs) were determined as described for SARS-CoV-2. 1, 12 WA-1 (SARS-CoV-2/USA-WA1/2020 EPI_ISL_404895) was obtained from BEI Resources while the, delta (hCoV19/USA/MD-HP05660/2021 EPI_ISL_2331507) and omicron (hCoV19/USA/MD-HP20874/2021 EPI_ISL_7160424) variants were isolated from COVID-19 patients at Johns Hopkins Hospital as previously described. 13 The nAb titer was calculated as the highest serum dilution that eliminated the cytopathic effect in 50% of the wells (NT50) and the area under the curve (AUC) was calculated using Graphpad Prism. The total plasma levels of antibodies against spike region S1 were measured using the ELISA Euroimmun assay as described. 14 Compared to the WA-1 isolate, we observed a 2-to 4-fold decrease in virus neutralization of delta and 20-to 40-fold neutralization decrease of omicron in all plasma samples (108 pre-delta/pre-omicron CCP units, the 20 post-delta COVID-19, post vaccination units and pre-delta/pre-omicron hyperimmunoglobulin) (Fig. 1) . The 108 research trial remnant units with Euroimmun AU over 3.5 have an 85% rate of positive virus neutralization for WA-1 and delta. However, Euroimmun over 10 AU was necessary to retain similar neutralization for omicron (Fig.1) . All of the post-delta COVID-19/post-vaccination donor plasma as well as the hyperimmunoglobulin, with Euroimmun AU over 10 effectively neutralized the WA-1 isolate, delta and omicron variants. As a benchmark for clinical effectiveness, the early treatment CCP trial, successful at preventing hospitalizations principally with unvaccinated ancestral virus, had more than 10% of pre-delta/pre-omicron 2020 prevaccination donor units or 2021 donor units with Euroimmun AU over 10 (Fig 1) . For all these clinical trial transfused units, the WA-1 neutralizing antibody geomean was 26 IU/mL 8 , while remnant 108 CCP plasma units had a geomean of 28 IU/mL. However, sorting to increasing Euroimmun AUs from greater than 3.5, to 7 and to 10 AUs, increased two-fold and ten-fold respectively WA-1, delta and omicron neutralization (Fig 2) . All of the post-delta COVID-19/post vaccination samples measured Euroimmun AU over 10, with WA-1 neutralization at geomean 1598 with comparable reduction in delta activity of two-fold and 20-fold with omicron (Fig. 2) . The pre-delta/pre-omicron hyperimmune globulin IgG showed a similar fold decrease in neutralization across the three isolates (Fig. 2) . The wider span of both high and low levels of neutralization antibodies in CCP may account for the larger decrease in delta activity compared to the narrower high range of virus neutralizations with the post-delta COVID-19/post-vaccination units. This data suggests that Euroimmun AU over 5 (~1.5X3.5) would be effective for delta and over 10 (~3X3.5) for omicron CCP therapy. Commercial serologic assays were adjusted approximately 1.5-fold by the FDA to be in the effective range for omicron. 9 The Euroimmun assay is one of many that can be utilized for EB-member of the FDA Blood Products Advisory Committee Appendix The members of the COVID-10 Serologic Studies Consortium (CSSC) are: Anne Arundel Medical Center Hopkins Bloomberg School of Public Health The Bliss Group The Next Practice Group (Colin Foster) Western Connecticut Health Network, Danbury Hospital Western Connecticut Health Network, Norwalk Hospital Sex, age, and hospitalization drive antibody responses in a COVID-19 convalescent plasma donor population Comparative Performance of Five Commercially Available Serologic Assays To Detect Antibodies to SARS-CoV-2 and Identify Individuals with High Neutralizing Titers Association between SARS-CoV-2 Neutralizing Antibodies and Commercial Serological Assays Considerable escape of SARS-CoV-2 Omicron to antibody neutralization Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift SARS-CoV-2 Omicron BA.2 Variant Evades Neutralization by Therapeutic Monoclonal Antibodies LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants Randomized Controlled Trial of Early Outpatient COVID-19 Treatment with High-Titer Convalescent Plasma. medRxiv Convalescent Plasma EUA Letter of Authorization 12282021 . FDA website Resistance of SARS-CoV-2 Omicron Variant to Convalescent and CoronaVac Vaccine Plasma SARS-CoV-2 Omicron Variant Neutralization in Serum from Vaccinated and Convalescent Persons An immunosuppressed Syrian golden hamster model for SARS-CoV infection A Quick Displacement of the SARS-CoV-2 variant Delta with Omicron: Unprecedented Spike in COVID-19 Cases Associated with Fewer Admissions and Comparable Upper Respiratory Viral Loads. medRxiv Clinical Validity of Serum Antibodies to SARS-CoV-2 : A Case-Control Study We gratefully acknowledge the generous contributions of the study participants and plasma donors who gave of their time and specimens. The study was funded principally by the U.S. Department of Defense's Joint Program Executive Office for Chemical, Biological, Radiological and Nuclear Defense (JPEO-CBRND), in