key: cord-0943341-vuepet6t authors: Bartelt, L. A.; Markmann, A. J.; Nelson, B. G.; Keys, J.; Root, H.; Henderson, H. I.; Kuruc, J.; Baker, C.; Bhowmik, D. R.; Hou, Y. J.; Premkumar, L.; Cornaby, C.; Schmitz, J.; Weiss, S.; Park, Y.; Baric, R. S.; de Silva, A. M.; Lachiewicz, A.; Napravnik, S.; van Duin, D.; Margolis, D. M. title: Outcomes of convalescent plasma with defined high- versus lower-neutralizing antibody titers against SARS-CoV-2 among hospitalized patients: CoronaVirus Inactivating Plasma (CoVIP), double-blind phase 2 study. date: 2022-05-02 journal: nan DOI: 10.1101/2022.04.29.22274387 sha: 842a0bf59333b5753521094c4381ea1b41984e1a doc_id: 943341 cord_uid: vuepet6t Background COVID-19 Convalescent Plasma (CCP) was an early and widely adopted putative therapy for severe COVID-19. Results from randomized control trials and observational studies have failed to demonstrate a clear therapeutic role for CCP for severe SARS-CoV-2 infection. Underlying these inconclusive findings is a broad heterogeneity in the concentrations of neutralizing antibodies (nAb) between different CCP donors. The present study was designed to evaluate nAb titer threshold for clinically effective CCP. Methods We conducted a double-blind, phase 2 study to evaluate the safety and effectiveness of nAb titer-defined CCP in adults admitted to an academic referral hospital. Patients positive on a SARS-CoV-2 nucleic acid amplification test and with symptoms for <10 days were eligible. Participants received either CCP with nAb titers [≥] 1:160-1:640 (standard titer group) or >1:640 (high titer group) in addition to standard of care treatments. Adverse events were contrasted by CCP titer. The primary clinical outcome was time to hospital discharge, with mortality and respiratory support evaluated as secondary outcomes. Findings Between August 28 and December 4, 2020, 316 participants were screened, 55 received CCP, with 41 and 14 receiving standard versus high titer CCP, respectively. Participants were a median of 61 years of age (IQR 52-67), 36% women, 25% Black and 33% Hispanic. Severe adverse events (SAE) ([≥]grade 3) occurred in 4 (29%) and 23 (56%) of participants in the high versus standard titer groups, respectively by day 28 (Risk Difference -0.28 [95% CI -0.56, 0.01]). There were no observed treatment-related AEs. By day 55, time to hospital discharge was shorter among participants receiving high versus standard titer, accounting for death as a competing event (hazard ratio 1.94 [95% CI 1.05, 3.58], Gray's p=0.02). Interpretation In this phase 2 trial in a high-risk population of patients admitted for Covid-19, we found earlier time to hospital discharge and lower occurrences of life-threatening SAEs among participants receiving CCP with nAb titers >1:640 compared with participants receiving CCP with lower nAb titer CCP. Though limited by a small study size these findings support further study of high-nAb titer CCP defined as >1:640 in the treatment of COVID-19. inconclusive findings is a broad heterogeneity in the concentrations of neutralizing antibodies 32 (nAb) between different CCP donors. The present study was designed to evaluate nAb titer 33 threshold for clinically effective CCP. 34 We conducted a double-blind, phase 2 study to evaluate the safety and effectiveness of nAb titer-36 defined CCP in adults admitted to an academic referral hospital. Patients positive on a SARS-37 CoV-2 nucleic acid amplification test and with symptoms for < 10 days were eligible. Participants 38 received either CCP with nAb titers ≥1:160-1:640 (standard titer group) or >1:640 (high titer 39 group) in addition to standard of care treatments. Adverse events were contrasted by CCP titer. 40 The primary clinical outcome was time to hospital discharge, with mortality and respiratory 41 support evaluated as secondary outcomes. 42 Findings 43 This phase 2 study showed that CCP with high nAb titer (>1:640) provided more rapid recovery 88 to hospital discharge and fewer COVID-19 attributable AEs than CCP with nAb titer between the 89 FDA-recommended minimum standard and 4-fold higher (≥1:160-1:640). The hazard ratio of time 90 to hospital discharge from baseline through day 55, accounting for death as a competing event, 91 contrasting patients receiving high versus standard CCP titer was 1.94 (95% CI 1.05-3.58). 92 Adjusted hazard ratios of high versus standard titer CCP receipt for time to hospital discharge were 93 consistent with the primary unadjusted findings. Mortality through 55 days was lower in the high 94 titer group, but with a wide confidence interval that did not reach statistical significance. 95 Our data that CCP with nAb >1:640 expedites recovery of patients admitted with COVID-19 97 compared with CCP with nAb ≥1:160-1:640 suggests that a threshhold of nAb ≥1:160 may be too 98 low to define CCP as 'high titer'. Analyses in larger CCP trials should consider full reporting of 99 nAb in CCP units administered at individual study participant level, and specifically whether CCP 100 contained nAb >1:640. Further investigation of CCP with nAb >1:640 is warranted given that and interpreting CCP clinical trials are heterogeneity in the source of the CCP product and wide 112 inter-individual variability in the breadth and potency of anti-SARS-CoV-2 neutralizing antibodies 113 (nAbs) and antibodies with non-neutralizing functions between different CCP donors. 6 Indeed, 114 secondary analyses in CONCOR-1 identified that antibody content and/or CCP supplier had a 115 significant effect on estimates of 30 day mortality and the need for ventilation among the CCP-116 treated participants. 4 Findings in large multicenter community-based observational studies suggest 117 that CCP with higher antibody levels is associated with a lower risk of in-hospital mortality, 7-9 and 118 more recently, use of high antibody titer CCP in outpatients reduced risk for hospitalization. 10 119 These findings led to emergency use authorization from the FDA and recommendations from 120 expert societies to use high antibody titer CCP as an oupatient therapeutic for high-risk individuals 121 when other therapies are not available. 122 Antibody-content is considered a major determinant of CCP safety and effectiveness, however, 123 there remains no standardized assay for distinguishing high antibody titer CCP with potential 124 clinical benefit from CCP without benefit or CCP that may be harmful. 4 For example, the FDA 125 initially recommended neutralizing antibody titers of >1:160 as qualification for CCP as treatment. 126 However, the limited ability to perform live viral neutralization assays prohibited most trials from 127 using this threshold. Rather, large trials like RECOVERY, qualified high titer CCP using an anti-128 SARS-CoV-2 spike IgG antibody ELISA (EUROIMMUN) with index value of > 6.0. 3 In contrast, 129 including IgG titers targeted to the spike protein receptor binding domain (RBD), correlate but 134 alone they are imperfect surrogates for precise functional viral neutralizing capability of CCP. 11,12 135 To circumvent the limitation of relying on a single antibody-binding assay as a measure of the 136 global anti-viral activity of a polyclonal therapy like CCP, we designed a study administering CCP 137 with pre-defined ranges of neutralizing antibody. Using an on-site SARS-CoV-2-WA1 viral 138 reporter neutralization assay also used in the development of mRNA vaccines, we precisely 139 defined the functional viral inhibitory properties of CCP. Beginning in April 2020, our medical 140 center began collecting locally sourced CCP for use in our hospital. 6 Here we report outcomes 141 from a pilot phase 2 double-blind study in patients admitted with severe COVID-19 using pre-142 treatment nAb titer-defined CCP from our donor cohort in two different ranges: FDA-minimum 143 recommend nAb titer of 1:160-1:640 (standard titer) and >1:640 (high titer). 144 Each participant was to receive two units of CCP (a total of ~400 -500 mL) within 48 hours of 172 randomization. The two units could be infused up to 24 hours apart. All eligible participants also 173 received institutional-guided standard-of-care. 174 Participants were randomly assigned in a 1:1 ratio to receive either high or standard titer CCP, 176 with randomization stratified by ABO blood group, using permuted block group randomization. 177 During the conduct of the study 13 participants randomized to receive high titer received standard 178 titer CCP because appropriate ABO-compatible high titer CCP was not available. The decision to 179 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2022. ; https://doi.org/10.1101/2022.04.29.22274387 doi: medRxiv preprint the apheresis diversion pouch was collected and transported on ice for fractionation into serum 202 and cellular components as approved by UNC IRB #20-1141. 203 Anti-SARS-CoV-2 antibody assays 204 Neutralizing antibody titers were measured using SARS-CoV-2-WA1 viral reporter 205 neutralization assay expressing a nano-luciferase gene, and was recovered using reverese genetics 206 as previously described. 14 Neutralization assays were performed as previously described. 6,11,14 207 This same assay has been used to assess mRNA vaccine effectiveness. 15 The minimum threshold 208 for CCP in the standard titer group was set at the initial FDA initial recommended titer of ≥1:160 209 (standard titer). The high nAb-titer minimum was set to at ≥ 4-fold difference higher than the 210 standard titer group, and to exceed the upper limit of viral neutralization plaque-assay based titers 211 reported at the time of study design (>1:640). 16 212 Binding antibody assays to SARS-CoV-2 spike protein receptor binding domain (RBD) and 213 nucleocapsid (N) IgG (Abbott Laboratories) were done as previously described. 6 Briefly, RBD 214 IgG, IgA and IgM end-point titers were obtained on heat-inactivated serum samples in an in-house 215 developed enzyme-linked immunosorbent assay format starting with a titer of 1:20. 11 Nuceocapsid 216 IgG levels were measured in the EUA approved Abbott SARS-CoV-2 IgG assay in a CLIA 217 certified laboratory using the Abbott Architect i2000SR immunoassay analyzer, further described 218 Recipients 220 Participants were monitored by hospital staff during and after each CCP infusion according to 221 standard hospital operating procedures. Clinical symptoms (vital signs and physical exam (as 222 documented by the treating teams), 8-point WHO Ordinal status, laboratory data, and adverse 223 event assessments were obtained on day 1, day 3 (+/-1), day 7 (+/-2), day 14 (+/-2), day 21 (+/-224 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2022. Outcomes 232 The primary safety endpoint was the cumulative incidence of serious adverse events (SAEs) at 233 study days 14 and 28 after the first CCP infusion. The primary clinical effectiveness endpoint was 234 the days to hospital discharge following the first dose of CCP. Exploratory clinical endpoints 235 included mortality, changes in clinical severity scores (e.g., WHO ordinal clinical status scale), 236 and days of supplemental oxygen, non-invasive ventilation/high-flow oxygen, and/or invasive 237 ventilation/ECMO requirement. All participants were followed until hospital discharge (day 55 238 post first infusion), with an additional 6 months of follow-up obtained through review of the 239 institutional electronic health record. 240 Given that 50% of the particpants randomized to the high titer group instead received standard 242 titer CCP, we performed an adjusted as-treated analysis, in effect treating the trial as an 243 observational study accounting for confounding. 17 We used standard statistics to describe patient 244 demographic and clinical characteristics at baseline (date of randomization / first unit of CCP). We 245 compared distributions of baseline characteristics by CCP titer received using Fisher's exact, 246 Pearson's chi-square and Kruskal-Wallis tests, as appropriate. We contrasted primary safety 247 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2022. received was compared using the log-rank test and hazard ratios were estimated using Cox 257 proportional hazards models. All hypothesis testing was two-sided. Statistical analyses were 258 performed using SAS software, version 9.4 (SAS Institute) and R software, version 4.0. 259 The funders of the study had no role in study design, data collection, data analysis, data 261 interpretation, or writing of the report. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2022. To our knowledge, this is the first report of the safety and effectiveness of pre-assigned CCP 327 defined as having nAb titer range of 1:160-1:640 versus more than 4-fold higher nAb titer 328 (>1:640). We used these titer range cutoffs based on the initial FDA-guidance that CCP be defined 329 as having nAb ≥1:160, the internal variability of the nAb assay being ~ 2-fold, and our published 330 findings that the >1:640 represented the top quartile of CCP donors in our program. 6 We found 331 that patients receiving high titer experienced accelerated time to recovery to hospital discharge, 332 and a trend towards lower mortality through 55 days post-infusion. Adverse events were common 333 in both groups, but were reflective of underlying pathologies attributable to COVID-19 and not 334 from the intervention. Despite a small sample size phase 2 study, unique strengths of our study 335 include pre-defined titer assays assignments using a WT nLuc SARS-CoV-2 viral neutralization 336 assay that is a direct measure of functionally neutralizing antibody and has been used in the 337 development FDA-authorized mRNA-vaccines, 15 and implementation after corticosteroids and 338 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2022. ; https://doi.org/10.1101/2022.04.29.22274387 doi: medRxiv preprint COVID-19 who had not yet developed a seroresponse. 18 In our exploratory analysis we also find 386 that earlier patient seroconversion is associated with better outcome, but in our study the most 387 advantageous outcomes were among those who also received the highest antibody content CCP. 388 These findings may indicate synergy between the nAb in CCP and the recipient pre-infusion nAb. 389 Further investigation is needed to better understand of the potential for polyclonal antibodies to 390 work additively or synergistically together, and whether polyclonal antibodies might decrease the 391 risk of treatment-emergent mutations during RBD-directed monoclonal antibody therapy that can 392 rapidly lead to antibody-resistant variants and subsequently prolonged viral shedding. 42 In 393 addition, a more complex mixture of neutralizing antibodies, such as CCP from vaccinated 394 individuals after Omicron breakthrough infection, might retain activity against variants of concern 395 like Omicron BA.1 and BA.2. that were not inhibited by several available commercial monoclonal 396 antibodies at the time of their emergence. 43 397 Our study has several limitations. First, like several prior CCP RCTs, we had difficulty reaching 398 target enrollment for each of our titer-defined groups. Our inability to identify enough donors with 399 high titer CCP resulted in 50% of the patients randomized to the high nAb titer group instead 400 receiving standard nAb titer CCP. We therefore used a standard observational study analytic 401 approach (ie. adjusted as-treated) considering a conventional intention-to-treat analysis 402 uninterpretable. 17 Since treatment assignment deviation was based solely on CCP availability and 403 investigators and treating providers remained blinded to treatment received, we did not observe 404 any confounding and our adjusted analyses were consistent to our crude results. Second, since we 405 could not adequately power a three-arm study, we were unable to include a CCP-free group. Third, 406 we are underpowered in this single-center study to reach a statistical conclusion for outcomes like 407 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2022. ; https://doi.org/10.1101/2022.04.29.22274387 doi: medRxiv preprint in-patient mortality. Fourth, since D614G was the most common variant in circulation during out 408 study, we cannot extrapolate our findings to other variants like Omicron BA.1 and BA.2. 409 As SARS-CoV-2 variants continue to emerge and spread, it is possible that CCP will remain an 410 alternative therapeutic should others be unavailable or ineffective against an emergent variant. We 411 suggest that a definition of high titer CCP exceeding >1:640 gives the greatest confidence in 412 benefit potential, and that future CCP trials deliberately use direct measures of the functional anti-413 SARS-CoV-2 properties to more precisely pre-assign CCP and avoid infusing low titer CCP that 414 is unlikely to have clinical beneft. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2022. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2022. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2022. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2022. ; https://doi.org/10.1101/2022.04.29.22274387 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2022. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2022. ; https://doi.org/10.1101/2022.04.29.22274387 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2022. ; https://doi.org/10.1101/2022.04.29.22274387 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2022. ; https://doi.org/10.1101/2022.04.29.22274387 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2022. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2022. Unadjusted mortalioty estimates by nAb titer . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2022. ; https://doi.org/10.1101/2022.04.29.22274387 doi: medRxiv preprint CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2022. ; https://doi.org/10.1101/2022.04.29.22274387 doi: medRxiv preprint :320) 5-10 ≥1:80 (1:10-≥1:80) 5-10 SOC=standard of care, FFP= fresh frozen plasma. ^a minimum nAb was required to qualify 624 CCP for the study. * nAb titers extrapolated from scatter plot Relative risks (95% CI) were independently calculated from published values using R studio. 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